Endometriosis is an estrogen-dependent disease that is characterized by the growth of endometrial tissue outside of the uterine cavity. The most common endometriotic lesions are ovarian endometrioma, peritoneal lesions, and deeply-infiltrating endometriosis. Ten percent of women in reproductive age are affected, a gross underestimate due to the delay in diagnosis and non-specific symptoms. The etiology of endometriosis is not well understood, making diagnosis difficult, and treatments suboptimal. Currently, laparoscopic surgery is the gold standard for diagnosis, however this method is invasive, costly, and physicians are often reluctant to send their patients to surgery without certainty of disease. It is therefore a research priority to identify a minimally-invasive biomarker for endometriosis. Over the years, the search for a biomarker has shifted from a single circulating biomarker, to a panel of circulating biomarkers, and finally to the advent of newer technologies. The studies of proteomics, genomics, phenomics, and metabolomics have shown some promise thus far. MicroRNAs, a discovery of genomics, are short, non-coding RNA strands that regulate mRNA expression by silencing or degrading the transcript. The dysregulation of miRNAs have been shown to contribute to the pathology of many gynecological conditions, and have shown to be dysregulated in endometriosis. To date however, results have been underwhelming due to differences in methodologies and failure to consider endometriosis as a heterogeneous disease. Three miRNAs were studied based on their prevalence in the literature (miR-9, -21, and -424), and three others (miR-10a, -10b, and -204) were measured based on their association with BDNF. In the current study, miR-204 expression was significantly lower (p=0.0016) in the eutopic endometrium of women with endometriosis compared to controls. Relative expression of miR-21, miR-424, and miR-10b differed significantly (p<0.05) across lesion types in women with exclusively endometriomas, peritoneal or deep-infiltrating lesions. Corresponding BDNF expression in the lesion types were inversely correlated to miRNA expression suggesting these miRNA regulate BDNF and are implicated in endometriosis pathology. Due to the findings that miRNAs are differentially expressed between endometriotic lesions, this study also suggests that, different lesion types are biochemically distinct. / Thesis / Master of Health Sciences (MSc)

Endometriosis is an estrogen dependent disease characterized by the growth of endometrial epithelium and stromal cells outside the uterine cavity. Lack of a clinical test results in a diagnostic delay of between 6-12 years. Recent studies suggest that miRNAs may be useful diagnostic tools; however, results remain equivocal. Use of different reference miRNA and definitions of control groups are factors postulated to contribute to the inconsistent findings in the literature. Serum samples were collected from women (n=53) undergoing laparoscopic surgery. Reference RNAs and symptomatic vs asymptomatic control groups were studied. Comparisons were made between cases and controls, controls and treated vs non-treated cases, and controls, endometriosis and adenomyosis. Data were compared by Mann Whitney U tests and Kruskal Wallis tests. A p value  0.05 was considered statistically significant. Our major finding was that reference RNA selection and categorization of patients influenced results. When using the most appropriate reference and comparing to asymptomatic controls, miR-9 was upregulated and miR-451a was downregulated in women with endometriosis. When using the most appropriate reference and comparing to symptomatic controls, miR-9 and 141 were downregulated in women with endometriosis. miR-9 and 141 were also downregulated in women receiving treatment. When using the most appropriate reference and comparing to women with adenomyosis, miR-451a, 20a and 122 were downregulated in women with endometriosis. While miRNA is the newest and has most promise as a diagnostic biomarker, miRNA expression results are extremely sensitive to multiple experimental variables. Literature continues to approach miRNA research using different definitions of control populations and different reference RNA. To replicate results and advance the search for miRNA as a diagnostic biomarker for endometriosis, a common methodology between labs will be necessary. / Thesis / Master of Science (MSc) / Endometriosis affects 10% of women within their reproductive lifespan. Since the gold standard for diagnosing endometriosis is laparoscopy, a diagnostic biomarker for endometriosis is of utmost importance. Currently there is no universally acceptable biomarker and circulating levels of miRNA appear to be promising but results have yet to be replicated. Replication of results may be dependent on reference RNA and definitions of control groups.

Nicht-codierende RNAs sind ein bedeutender Bestandteil genregulatorischer Prozesse. Ihre Fehlregulierung wird mit zellulärer Dysfunktion und der Entstehung von Krankheiten in Zusammenhang gebracht. Ziel dieser Arbeit war die Entwicklung verschiedener Testsysteme zur Untersuchung nicht codierender RNAs mit dem Schwerpunkt microRNA (miRNA), precursor miRNA (pre-miRNA) und circular RNA (circRNA). Für eine Zyklisierung und Funktionalisierung von circRNA mittels Cu-katalysierter Click-Chemie zur Identifizierung zellulärer Interaktionspartner und zugehöriger Wirkmechanismen wurden die Termini linearer RNA-Template modifiziert. Mit Hilfe enzymatischer Techniken wie Transkription und Ligation konnte in vitro die Inkorporation Azid- und Alkin-funktionalisierter Nukleotid-Bausteine am 5‘- und 3‘-Terminus gezeigt werden. Zur Untersuchung der miRNA-Reifung in cellulo wurde die pre-miRNA-134 unter Verwendung chemo-enzymatischer Methoden mit einem Fluorophor/Quencher-Paar an den Termini ausgestattet. Durch intrazelluläre Reifung der gelabelten pre-miRNA mit einhergehender Fluoreszenzfreisetzung sollte die Visualisierung und damit die Lokalisierung des miRNA-Reifungsortes innerhalb von Neuronen realisiert werden. Zudem gelang die Entwicklung eines auf branched rolling-circle amplification (BRCA) basierenden Argonaute2(Ago2)-vermittelten Spaltungsassays. Ein Enzymkomplex aus rekombinantem, humanem Ago2 und der miRNA miR 122, genannt minimal RISC, wurde dabei zur Substrat-Spaltung eingesetzt. Zur Etablierung des BRCA-basierenden Ago2-vermittelten Spaltungsassays als Screening-Tool für die Identifizierung potentieller Inhibitoren der mRNA-Spaltung wurden exemplarisch sechs Testsubstanzen aus der Gruppe der Aminoglykoside untersucht. Der BRCA-basierende Ago2-vermittelte Spaltungsassay stellte eine einfache und zuverlässige Detektionsmethode dar, der die Untersuchung einer größeren Probenzahl mit geringem Aufwand und ohne Verwendung von fluorogen gelabeltem Substrat ermöglichte. / Non-coding RNAs are an important factor in gene regulation in which their deregulation is associated with cellular dysfunction and disease. Here, the development of different test systems for the investigation of non-coding RNAs, namely microRNA (miRNA), precursor miRNA (pre-miRNA), and circular RNA (circRNA), was on focus. In order to circularize and functionalize circRNA with the purpose of identifying cellular interaction partners and possible mechanisms of action, 5‘- and 3‘-terminal modifications were added to a linear RNA template. This was accomplished by using azide- and alkyne-functionalized nucleotides which were incorporated by enzymatic approaches like transcription and ligation to be followed by Cu-catalyzed click chemistry for circularization. For investigating miRNA maturation in neuronal cells, pre-miR-134 was modified by chemo-enzymatic approach with fluorophore and quencher at its 5‘ and 3‘ ends, respectively. Intracellular maturation of labeled pre-miRNA would produce a fluorescent signal upon cleavage, thus enabling visualization and localization of miRNA maturation in neuronal cells. Furthermore, the development of Ago2-mediated mRNA cleavage assay based on branched rolling-circle amplification (BRCA) was accomplished. A complex of recombinant human Ago2 and miRNA miR-122, called minimal RISC, was used for substrate cleavage. To establish this assay as adequate screening method for identifying potential inhibitors of mRNA cleavage, a group of six aminoglycosides was tested. The BRCA-based Ago2-mediated cleavage assay showed to be a simple and reliable detection method and screening tool for small molecule binders with little effort and without fluorescent labeling of substrate.

The circadian system in mammals is arranged as a hierarchical network of oscillators, with the master pacemaker of circadian rhythms located in the suprachiasmatic nuclei (SCN) of the hypothalamus and peripheral oscillators in most other organ and tissue systems of the body. The molecular machinery responsible for generating circadian rhythms is composed of interlocked transcriptional-translational feedback loops with the gene Brain Muscle Arnt-like 1 (Bmal1) functioning as a core positive regulator. Using the mouse, Mus musculus as a model system, we studied the post-transcriptional mechanisms regulating Bmal1 expression in the SCN pacemaker and in peripheral oscillators. Target prediction algorithms were used to identify microRNAs (miRNAs) predicted to target Bmal1. We profiled the temporal expression of miR-142-3p in the mouse SCN in vivo and in an immortalized SCN cell line and observed robust circadian rhythms in its expression in the SCN. Following luciferase-reporter and site-directed mutagenesis analyses, we identified miR-142-3p as a bona-fide post-transcriptional repressor of Bmal1. The temporal expression of potential Bmal1-targeting miRNAs was also examined in the circulation in mouse serum. In mice housed in a light-dark cycle, diurnal oscillations were observed in serum levels of miR-152 and miR-494, but not miR-142-3p expression. Luciferase reporter studies indicated that miR-494, both independently and synergistically with miR-142-3p, repressed the Bmal1 3′ UTR. Overexpression of these miRNAs disrupted ensemble circadian rhythms of PER2::LUCIFERASE activity in cultured fibroblasts. Overexpression of the miRNAs also increased their extracellular levels and their intracellular accumulation in recipient cultures exposed to conditioned medium. Furthermore, inhibition of exocytosis and endocytosis affected ensemble circadian rhythms in cultured fibroblasts. The results thus implicate miR-142-3p and miR-494 in the regulation of Bmal1 expression in the SCN and peripheral oscillators and suggest that miRNAs may function as both, intracellular and extracellular (cis- and trans- acting) signals, modulating the core clock mechanism in the SCN and in fine-tuning the synchronization of circadian rhythmicity between cell-autonomous oscillators in the periphery.

Introdução: A ereção peniana é um processo neurovascular complexo iniciado através de estimulação sexual, que podem ser fatores físicos ou psicológicos com atuação de substâncias no endotélio vascular resultando no ato sexual satisfatório. Diversos fatores podem interferir de forma negativa no mecanismo fisiológico da ereção peniana, consequentemente, levando a disfunção erétil, tais fatores como hipertensão arterial sistêmica, diabetes mellitus, tabagismo e alcoolismo. O etanol é uma das substâncias mais consumidas nas diferentes sociedades, tornando-se, assim, alvo de inúmeros estudos que visam conhecer sobre sua toxicidade. Apesar da grande associação entre o consumo de álcool e o aumento da incidência da disfunção erétil, os mecanismos moleculares envolvidos ainda são poucos conhecidos. Devido a abundância, os microRNAs tem sido descrito envolvidos em diversas funções fisiológicas dos processos celulares, e além disso estão envolvidos em muitos processos fisiopatológicos, como, por exemplo, a disfunção erétil. Dentre os processos celulares, os microRNAs exercem funções anti ou pró apoptóticas. Embora a conexão dos microRNAs a apoptose esteja bem estabelecida, sua função em resposta a apoptose induzida pelo etanol permanece em grande parte desconhecida. Objetivos: Avaliar o mecanismo de apoptose, pela expressão de AIF e PARP, assim como dos seus microRNAs reguladores: miR-145, miR-210 e miR-486, nos corpos cavernosos de ratos submetidos a modelo de \"alcoolismo semivoluntário\". Material e métodos: Foram utilizados 12 ratos Wistar divididos em dois grupos: controle (C) e tratado com etanol (A) a 20% durante sete semanas. As amostras dos corpos cavernosos foram preparadas para o estudo morfométrico pela coloração de tricrômico de Masson, para o estudo da expressão protéica de AIF e PARP por imunohistoquímica e para o estudo da expressão gênica no tecido cavernoso do miR- 145, miR-210 e do miR-486, por PCR em tempo real. Resultados: A análise imuhistoquímica mostrou pouca marcação positiva nuclear para a proteína PARP e AIF nos corpos cavernosos dos animais dos grupos controle e tratado com etanol. Após análise da expressão dos microRNAs miR-145, -210 e -486, nos 12 animais estudados, não foram encontrados resultados com diferença estatística significativa entre os grupos controle e alcoolizado. Conclusões: A expressão gênica do miRNA -145 e miRNA-486 foi maior nos animais do grupo tratado com etanol quando comparado aos animais do grupo controle; A expressão gênica do miRNA - 210 foi maior nos animais do grupo controle quando comparado aos animais do grupo tratado com etanol; Foi observada correlação entre a expressão protéica de PARP e AIF e do miRNA -210, ambos com maior marcação positiva no grupo controle. / Introduction: Penile erection is a complex neurovascular process initiated through sexual stimulation, which can be physical or psychological factors with action of substances in the vascular endothelium resulting in the satisfactory sexual act. Several factors can negatively interfere in the physiological mechanism of penile erection, consequently leading to erectile dysfunction, such factors as systemic arterial hypertension, diabetes mellitus, smoking and alcoholism. Ethanol is one of the most consumed substances in different societies, becoming, therefore, the target of numerous studies that aim to know about its toxicity. Despite the large association between alcohol consumption and the increased incidence of erectile dysfunction, the molecular mechanisms involved are still poorly understood. Due to abundance, microRNAs have been described involved in various physiological functions of cellular processes, and in addition are involved in many pathophysiological processes, such as erectile dysfunction. Among the cellular processes microRNAs have anti-apoptotic or pro functions. Although the connection of the microRNAs to apoptosis is well established, their function in response to ethanol-induced apoptosis remains largely unknown. Objectives: To evaluate the mechanism of apoptosis, by the expression of AIF and PARP, as well as of its regulatory microRNAs: miR-145, miR-210 and miR-486, in the cavernous bodies of rats submitted to a model of \"semi-voluntary alcoholism\". Material and methods: 12 Wistar rats were divided into two groups: Control (C) and treated with ethanol (A) diluted 20% for seven weeks. Samples of the corpus cavernosum were prepared for morphometric study by Masson\'s trichrome stain, study for protein expression of CASPASE 3 (pro-apoptotic) by immunohistochemistry and study for gene expression of miRNA-21 (anti-apoptotic) in serum and the cavernous tissue. Results: The immunohistochemical analysis showed little positive nuclear marking for the PARP and AIF protein in the corpus cavernosum of the control and ethanol treated groups. After analysis of miR-145, -210 and -486 microRNA expression in the 12 animals studied, no statistically significant differences were found between the control and alcoholized groups. Conclusions: The gene expression of miRNA-145 and miRNA-486 was higher in the animals of the ethanol treated group when compared to the animals in the control group; Gene expression of miRNA - 210 was higher in the control group than in the animals treated with ethanol; A correlation was observed between the protein expression of PARP and AIF and of the -210 miRNA, both with greater positive marking in the control group.

A artrite reumatoide (AR) é uma doença crônica autoimune que afeta as articulações e causa uma persistente inflamação sinovial e destruição da cartilagem e osso. A artrite induzida por pristane (PIA) em camundongos é um modelo experimental utilizado em muitos trabalhos, pois se assemelha à artrite reumatoide. MicroRNAs (miRNA) têm sido bastante estudados por sua participação no desenvolvimento da artrite. As linhagens de camundongos AIRmax e AIRmin, diferem quanto a susceptibilidade/resistência à PIA. Nós analisamos o perfil de expressão gênica de mRNA e miRNAs das células peritneais dessas linhagens e avaliamos sua participação no desenvolvimento da PIA. A linhagem AIRmax mostrou uma maior modulação de genes (2025) com relação aos AIRmin (1043) no início dos sintomas. Os miRNAs 132-3p/212-3p e 130b-3p foram, os miRNAs mais significativamente modulados nos animais sensíveis e mostraram correlações negativas com alguns de seus genes alvos preditos. Nosso estudo mostrou que a expressão de mRNAs e miRNAs é modificada nas linhagens AIRmax e AIRmin durante a PIA. / Rheumatoid arthritis is a chronic autoimmune disease that affects joints and it is characterized by synovial inflammation and articular cartilage and bone destruction. Pristane-induced arthritis (PIA) in mice is an experimental model that has been used in many studies, since it resembles rheumatoid arthritis. MiRNAs has been extensively studied in the development of arthritis. AIRmax and AIRmin lines, differ in their susceptibility/resistance to PIA. We analyzed the miRNA and gene expression profile of mRNA in peritoneal cells of these lines in order to evaluate their involvement in PIA development. AIRmax mice showed a high gene modulation (2025) than AIRmin mice (1043) at the onset of disease symptoms. The 132-3p/212-3p and 130b-3p miRNAs were the most significant in susceptible animals showing negative correlations with some of their predicted target genes. Our study showed that the global gene and miRNA expressions are modified in the peritoneal cells of AIRmax and AIRmin lines during pristane-induced arthritis.

O objetivo do presente estudo foi comparar o efeito da idade de doadores de oócitos sobre os parâmetros reprodutivos, a taxa de recuperação e qualidade de oócitos após ICSI utilizando sêmen sexado. Éguas Pôneis de Polo (n = 79) e Puro-Sangue (n = 23) doadoras de oócitos (400-600kg e 3-29 anos) foram utilizados durante as estações reprodutivas de 2009/2010 e 2011 no hemisfério Sul (San Luis, Argentina) e norte (Kentucky, EUA), respectivamente. As éguas foram divididas em três categorias experimentais: Jovens (YM: 3-10 anos), Meia Idade (MA: 11-17a) e Idosas (OM&ge;18a). Um total de 326 oócitos recuperados in vivo a partir de folículos pré-ovulatórios (n=279) e folículos imaturos em crescimento (n=47). Desses, 224 oócitos foram recuperados, classificado e dirigido a um programa comercial ICSI com Pôneis de Polo (primeira estação). Durante a segunda estação, 57 oócitos foram recuperados a partir de folículos pré-ovulatórios e 47 oócitos de folículos em crescimento e congelados em nitrogênio líquido para ser usado posteriormente em outro estudo. Os pontos experimentais avaliados foram: intervalos (dias) entre a aspiração ou a ovulação (AS/OV) ao PGF, PGF ao GnRH (Dia 0=GnRH), AS/OV ao Dia 0, e AS/OV ao Dia&#43;1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D&#43;1, e a taxa de crescimento folicular. O número total de aspirações, o número de folículos aspirados e oócitos por aspiração e/ou folículo, o grau de expansão das células do cúmulos, a presença e qualidade do corpo polar (PB), o volume ooplasma, o intervalo de GnRH a aspiração, a GnRH a ICSI, e a taxa de clivagem (CR) depois da ICSI também foram avaliados. Foram observadas diferenças significativas entre as três categorias experimentais (YM, MA e OM) para: intervalos (dias) entre PGF-GnRH, AS/OV-GnRH, AS/OV-Day 1, edema uterino no Dia 0, CT em Dia 1, MdF1 no D0 e D&#43;1 e volume de ooplasma. A taxa de recuperação in vivo (RR) não foi afetada pela idade (média de 102%, 85% e 73,4% de oócitos recuperados por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as categorias experimentais. Em conclusão, um efeito de envelhecimento pode ser observado em vários parâmetros reprodutivos em éguas. Os ovócitos no presente estudo tiveram um menor volume de ooplasma para OM comparados com YM, mas isso não foi eficaz em predizer a viabilidade dos oócitos ou o potencial para o desenvolvimento para a avaliação da taxa de clivagem após ICSI utilizando sêmen sexado / The aim of the present study was to compare the effect of oocyte donors` age on reproductive parameters, recovery rate and quality of oocytes after ICSI using sexed semen. Polo Ponies (n=79) and Thoroughbred (n=23) oocyte donor mares (400-600kg and 3-29 years) were used during the 2009/2010 and 2011 reproductive seasons in the Southern (San Luis, Argentina) and in the Northern hemispheres (Kentucky, USA), respectively. Mares were divided into three experimental categories: Young Mares (YM: 3-10&#1059;), Middle Age Mares (MA: 11-17&#1059;) and Old Mares (OM18&#1059;). A total of 326 oocytes were recovered in vivo from pre-ovulatory follicles (n=279) and immature-growing follicles (n=47). From those, 224 oocytes were recovered, sorted and directed to a commercial ICSI program with Polo Ponies (first season). During the study`s second season, 57 oocytes were recovered from pre-ovulatory follicles and 47 oocytes from growing follicles and frozen in liquid nitrogen to be used in another study. The evaluated experimental end-points were: Intervals (days) between aspiration or ovulation (AS/OV) to PGF, PGF to GnRH (Day 0=GnRH), AS/OV to Day 0, and AS/OV to Day &#43; 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D&#43;1, and follicular growth rate. Total number of aspirations, number of follicles aspirated and oocytes recovered per aspiration and/or follicle, the degree of cumulus cell expansion, the presence and quality of polar body (PB), the ooplasm volume, the interval from GnRH to aspiration, GnRH to ICSI, and the cleavage rate (CR) after ICSI were also evaluated. Significant differences between the three experimental categories (YM, MA and OM) were observed for: intervals (days) between PGF-GnRH, AS/OV-GnRH, AS/OV-Day &#43;1, uterine edema on Day 0, CT on Day &#43;1, MdF1 on D0 and D&#43;1 and ooplasm volume. The in vivo recovery rate (RR) was not affected by age (average of 102%, 85% and 73.4% of oocyte recovered per cycle, per F1 and per follicle, respectively; P>0.05). The CR did not differ among experimental categories. In conclusion, an effect of aging could be observed in several reproductive parameters in mares. The oocytes in the present study had a smaller ooplasm volume for OM compared to YM, but this was not effective in predicting oocyte viability or the potential to development through evaluation of the cleavage rate after ICSI using sexed semen

Die arbuskuläre Mykorrhiza ist die wahrscheinlich älteste Form der Wurzelsymbiosen zwischen Pflanzen und Pilzen und hat sich vor 420 Millionen Jahren entwickelt. In dieser Symbiose, die zwischen nahezu allen Landpflanzen und Pilzen des Reiches Glomeromycota ausgebildet wird, versorgt der Pilz die Pflanze mit Nährstoffen, wobei die verbesserte Versorgung mit Phosphat für die Pflanze sicher den größten Vorteil darstellt. Im Gegenzug erhält der Pilz Zucker, welche die Pflanze aus der Photosynthese bereitstellt. Zu hohe Phosphatkonzentrationen im Boden oder Dünger führen allerdings zu einer Verringerung in der Ausprägung der arbuskulären Mykorrhiza. Diese Unterdrückung der Symbiose wird nicht durch eine lokale Reaktion der Wurzeln ausgelöst, sondern in erster Linie durch einen hohen Phosphatgehalt im Pflanzenspross. Somit handelt es sich also um eine systemische, also dem Gesamtsystem „Pflanze“ betreffende Antwort. Die molekularen Mechanismen dieser Anpassung sind noch wenig bekannt und sind vor allem für die Agrarwirtschaft von besonderem Interesse. Eine Mikro-RNA (miRNA) des bereits bekannten Phosphathomöostasesignalwegs (PHR1-miRNA399-PHO2 Signalweg) akkumuliert verstärkt in mykorrhizierten Wurzeln. Das deutet daraufhin, dass dieser Signalweg und diese miRNA eine wichtige Rolle in der Regulation der arbuskulären Mykorrhiza spielen. Ziel dieser Studie war es neue Einblicke in die molekularen Mechanismen, die zur Unterdrückung der arbuskulären Mykorrhiza bei hohen Phosphatkonzentrationen führen, zu gewinnen. Dabei sollte der Einfluss von PHO2, sowie von miRNAs in dieser Symbiose genauer untersucht werden. Ein funktionelles Ortholog von PHO2, MtPho2, wurde in der Pflanze Medicago truncatula identifiziert. MtPho2-Mutanten, welche nicht mehr in der Lage waren ein funktionales PHO2 Protein zu exprimieren, zeigten schnellere Kolonisierung durch den AM-Pilz. Jedoch wurde auch in den mtpho2-Mutanten die Symbiose durch hohe Phosphatkonzentrationen unterdrückt. Dies bedeutet, dass PHO2 und somit der PHR1-miRNA399-PHO2 Signalweg eine wichtige Funktion während der fortschreitenden Kolonisierung der Wurzel durch den Pilz hat, aber und weitere Mechanismen in der Unterdückung der Symbiose bei hohen Phosphatkonzentrationen beteiligt sein müssen. Die Analyse von Transkriptionsprofilen von Spross- und Wurzeln mittels Microarrays zeigte, dass die Unterdrückung der AM Symbiose durch hohe Phosphatkonzentrationen möglicherweise auf eine Unterdrückung der Expression einer Reihe symbiosespezifischer Gene im Spross der Pflanze beruht. Um die Rolle weiterer miRNA in der AM Symbiose zu untersuchen, wurden mittels einer Hochdurchsatz-Sequenzierung 243 neue und 181 aus anderen Pflanzen bekannte miRNAs in M. truncatula entdeckt. Zwei dieser miRNAs, miR5229 und miR160f*, sind ausschließlich während der arbuskulären Mykorrhiza zu finden und weitere miRNAs werden während dieser Symbiose verstärkt gebildet. Interessanterweise führen einige dieser miRNAs zum Abbau von Transkripten, die eine wichtige Funktion in der arbuskulären Mykorrhiza und Wurzelknöllchensymbiose besitzen. Die Ergebnisse dieser Studie liefern eine neue Grundlage für die Untersuchung von regulatorischen Netzwerken, die zur zellulären Umprogrammierung während der Interaktion zwischen Pflanzen und arbuskulären Mykorrhiza-Pilzen bei verschiedenen Phosphatbedingungen führen. / AM symbiosis has a positive influence on plant P-nutrition and growth, but little is known about the molecular mechanism of the symbiosis adaptation to different phosphate conditions. The recently described induction of several pri-miR399 transcripts in mycorrhizal shoots and subsequent accumulation of mature miR399 in mycorrhizal roots indicates that local PHO2 expression must be controlled during symbiosis, presumably in order to sustain AM symbiosis development, in spite of locally increased Pi-concentration. A reverse genetic approach used in this study demonstrated that PHO2 and thus the PHR1-miR399-PHO2 signaling pathway, is involved in certain stages of progressive root colonization. In addition, a transcriptomic approach using a split-root system provided a comprehensive insight into the systemic transcriptional changes in mycorrhizal roots and shoots of M. truncatula in response to high phosphate conditions. With regard to the transcriptional responses of the root system, the results indicate that, although the colonization is drastically reduced, AM symbiosis is still functional at high Pi concentrations and might still be beneficial to the plant. Additionally, the data suggest that a specific root-borne mycorrhizal signal systemically induces protein synthesis, amino acid metabolism and photosynthesis at low Pi conditions, which is abolished at high Pi conditions. MiRNAs, such as miR399, are involved in long-distance signaling and are therefore potential systemic signals involved in AM symbiosis. A deep-sequencing approach identified 243 novel miRNAs in the root tissue of M. truncatula. Read-count analysis, qRT-PCR measurements and in situ hybridizations clearly indicated a regulation of miR5229a/b, miR5204, miR160f*, miR160c, miR169 and miR169d*/l*/m*/e.2* during arbuscular mycorrhizal symbiosis. Moreover, miR5204* represses a GRAS TF, which is specifically transcribed in mycorrhizal roots. Since miR5204* is induced by high Pi it might represent a further Pi status-mediating signal beside miR399. This study provides additional evidence that MtNsp2, a key regulator of symbiosis-signaling, is regulated and presumably spatially restricted by miR171h cleavage. In summary, a repression of mycorrhizal root colonization at high phosphate status is most likely due to a repression of the phosphate starvation responses and the loss of beneficial responses in mycorrhizal shoots. These findings provide a new basis for investigating the regulatory network leading to cellular reprogramming during interaction between plants, arbuscular mycorrhizal fungi and different phosphate conditions.

O objetivo do presente estudo foi comparar o efeito da idade de doadores de oócitos sobre os parâmetros reprodutivos, a taxa de recuperação e qualidade de oócitos após ICSI utilizando sêmen sexado. Éguas Pôneis de Polo (n = 79) e Puro-Sangue (n = 23) doadoras de oócitos (400-600kg e 3-29 anos) foram utilizados durante as estações reprodutivas de 2009/2010 e 2011 no hemisfério Sul (San Luis, Argentina) e norte (Kentucky, EUA), respectivamente. As éguas foram divididas em três categorias experimentais: Jovens (YM: 3-10 anos), Meia Idade (MA: 11-17a) e Idosas (OM&ge;18a). Um total de 326 oócitos recuperados in vivo a partir de folículos pré-ovulatórios (n=279) e folículos imaturos em crescimento (n=47). Desses, 224 oócitos foram recuperados, classificado e dirigido a um programa comercial ICSI com Pôneis de Polo (primeira estação). Durante a segunda estação, 57 oócitos foram recuperados a partir de folículos pré-ovulatórios e 47 oócitos de folículos em crescimento e congelados em nitrogênio líquido para ser usado posteriormente em outro estudo. Os pontos experimentais avaliados foram: intervalos (dias) entre a aspiração ou a ovulação (AS/OV) ao PGF, PGF ao GnRH (Dia 0=GnRH), AS/OV ao Dia 0, e AS/OV ao Dia&#43;1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D&#43;1, e a taxa de crescimento folicular. O número total de aspirações, o número de folículos aspirados e oócitos por aspiração e/ou folículo, o grau de expansão das células do cúmulos, a presença e qualidade do corpo polar (PB), o volume ooplasma, o intervalo de GnRH a aspiração, a GnRH a ICSI, e a taxa de clivagem (CR) depois da ICSI também foram avaliados. Foram observadas diferenças significativas entre as três categorias experimentais (YM, MA e OM) para: intervalos (dias) entre PGF-GnRH, AS/OV-GnRH, AS/OV-Day 1, edema uterino no Dia 0, CT em Dia 1, MdF1 no D0 e D&#43;1 e volume de ooplasma. A taxa de recuperação in vivo (RR) não foi afetada pela idade (média de 102%, 85% e 73,4% de oócitos recuperados por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as categorias experimentais. Em conclusão, um efeito de envelhecimento pode ser observado em vários parâmetros reprodutivos em éguas. Os ovócitos no presente estudo tiveram um menor volume de ooplasma para OM comparados com YM, mas isso não foi eficaz em predizer a viabilidade dos oócitos ou o potencial para o desenvolvimento para a avaliação da taxa de clivagem após ICSI utilizando sêmen sexado / The aim of the present study was to compare the effect of oocyte donors` age on reproductive parameters, recovery rate and quality of oocytes after ICSI using sexed semen. Polo Ponies (n=79) and Thoroughbred (n=23) oocyte donor mares (400-600kg and 3-29 years) were used during the 2009/2010 and 2011 reproductive seasons in the Southern (San Luis, Argentina) and in the Northern hemispheres (Kentucky, USA), respectively. Mares were divided into three experimental categories: Young Mares (YM: 3-10&#1059;), Middle Age Mares (MA: 11-17&#1059;) and Old Mares (OM18&#1059;). A total of 326 oocytes were recovered in vivo from pre-ovulatory follicles (n=279) and immature-growing follicles (n=47). From those, 224 oocytes were recovered, sorted and directed to a commercial ICSI program with Polo Ponies (first season). During the study`s second season, 57 oocytes were recovered from pre-ovulatory follicles and 47 oocytes from growing follicles and frozen in liquid nitrogen to be used in another study. The evaluated experimental end-points were: Intervals (days) between aspiration or ovulation (AS/OV) to PGF, PGF to GnRH (Day 0=GnRH), AS/OV to Day 0, and AS/OV to Day &#43; 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D&#43;1, and follicular growth rate. Total number of aspirations, number of follicles aspirated and oocytes recovered per aspiration and/or follicle, the degree of cumulus cell expansion, the presence and quality of polar body (PB), the ooplasm volume, the interval from GnRH to aspiration, GnRH to ICSI, and the cleavage rate (CR) after ICSI were also evaluated. Significant differences between the three experimental categories (YM, MA and OM) were observed for: intervals (days) between PGF-GnRH, AS/OV-GnRH, AS/OV-Day &#43;1, uterine edema on Day 0, CT on Day &#43;1, MdF1 on D0 and D&#43;1 and ooplasm volume. The in vivo recovery rate (RR) was not affected by age (average of 102%, 85% and 73.4% of oocyte recovered per cycle, per F1 and per follicle, respectively; P>0.05). The CR did not differ among experimental categories. In conclusion, an effect of aging could be observed in several reproductive parameters in mares. The oocytes in the present study had a smaller ooplasm volume for OM compared to YM, but this was not effective in predicting oocyte viability or the potential to development through evaluation of the cleavage rate after ICSI using sexed semen

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.