O presente estudo discute o papel dos miRNAs na fisiopatologia molecular de Esclerose Múltipla Recorrente Remitente (EMRR). O estudo demonstrou que em linfócitos T CD4+ de pacientes com EMRR em surto ocorre a diminuição da expressão do miR-15a e do miR16-1 em contraposição ao aumento de seu gene alvo BCL-2, um importante gene regulador da apoptose. Esses achados sugerem a participação desses miRNAs no controle da apoptose na EM. Para explorar essa associação, foi analisado a expressão global de miRNAs nas subpopulações de linfócitos T de pacientes com EMRR no estágio de remissão. O resultado dessa análise determinou de forma inédita o aumento significativo da expressão de 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) nos linfócitos T CD8+ de memória central. A análise in silico dos alvos desses miRNAs indicou que três vias canônicas, relacionadas à ativação da apoptose, eram enriquecidas com alvos preditos e validados experimentalmente desses miRNAs. Desse modo sugerimos a forte relação desses miRNAs no controle da apoptose nos linfócitos T dos pacientes com EMRR. A fim de aprofundar nossos estudos, selecionamos os miRNAs miR-21 e miR-24, para a realização de experimentos funcionais in vivo. Foi verificada a indução da expressão miR-21 somente nos linfócitos T CD4+ de modelo experimental da EM. Adicionalmente, experimentos in vitro demonstraram que a expressão do miR-21 e restrita as populações de células Th2 e Th17. Nesse caso, miR-21 parece ser regulado pelo fator de transcrição STAT3, sugerindo assim que o aumento da expressão do miR-21 verificada no modelo animal possa estar relacionada com a presença de linfócitos T CD4+ de perfil Th17 nesse tecido. Em resumo, o conjunto desses resultados demonstra a relevância dos miRNAs na fisiopatologia da EMRR, principalmente no controle da apoptose. / This study discusses the role of miRNA in the Molecular Pathophysiology of Relapse Remitting Multiple Sclerosis (RRMS). The study has shown that CD4+ lymphocytes from relapsed RRMS patients had lower expression of miR15a and miR-16-1 in contraposition of higher expression of the target gene BCL-2, a key regulator of apoptosis. These findings suggest the role of those on the control of apoptosis in MS. In order to explore this association, the global expression of miRNAs was analysed in T lymphocyte subpopulations from remission RRMS patients. The result of this analysis has demonstrated for the first time a significant higher expression of 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) in central memory T CD8+ lymphocytes. In silico analysis of the miRNAs targets indicates that three canonical pathways related to the activation of apoptosis were enriched with predicted and experimental validated gene targets for those miRNAs. In this way, we suggest the strong relation of these miRNAs in the control of apoptosis in the lymphocytes from RRMS patients. In order to intensify our studies we selected miR-21 and miR-24 to perform in vivo functional experiments. It was verified miR-21 induction only in T CD4+ lymphocytes from MS animal model. Additionally, in vitro experiments have demonstrated that miR-21 expression was restricted to Th2 and Th17 cell populations. In this way, miR-21 seems to be regulated by the STAT3 transcription factor, thereby suggesting that the increase of miR-21 expression observed in vivo could be related with Th17 CD4+ present in this tissue. In summary, this set of results showed the relevance of miRNAs in the RRMS pathophysiology, mainly in the control of apoptosis.

No description available.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_BiologiaMolecularAplicada.pdf: 8918060 bytes, checksum: d929a519e6a827337140388ee9beb358 (MD5) Previous issue date: 2016-09-02 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hanseníase é causada pelo Mycobacterium leprae e os indivíduos acometidos pela hanseníase podem ser classificados, em Paucibacilares e Multibacilares. Alternativamente, segundo Ridley-Jopling (1966), com base em critérios clínicos e imuno-hitológicos em outros dois pólos: (i) o pólo Tuberculóide (TT); e (ii) pólo Lepromatoso (LL), e seus intermediários. Independente de sua classificação, este espectro parecer ser inflluenciado por moléculas moduladoras da resposta imune, como os genes que codificam estes mediadores, e por um grupo de pequenos RNAs (microRNAs) que são responsáveis pela regulação destes genes, portanto essas investigações podem adensar o conhecimento sobre o mecanismo de resposta ao processo infecsioso, assim como possibilitar a identificação de novos biomarcadores no auxilio ao diagnóstico da hanseníase. O objetivo foi investigar oito polimorfismos do tipo INDEL nos genes CYP19A1, NFKβ1, IL1α, CASP8, UGT1A1, PAR1, CYP2E1, e IL4, para identificar possíveis marcadores de susceptibilidade e a influência da ancestria genética neste risco, além disso foi realizado o primeiro miRnoma da hanseníase por sequenciamento massivo em plataforma de alto desempenho, afim de elucidar o perfil epigenético presente na hanseníase. Nosso estudo revelou que os genes NFΚβ1, CASP8, PAR1 e IL4, são potenciais marcadores de susceptibilidade para a hanseníase, enquanto que NFΚβ1, CASP8, PAR1 e CYP19A1 são potenciais marcadores da forma clínica multibacilar. Adicionalmente, a análise da ancestralidade genômica mostrou que a contribuição Européia elevou o risco ao desenvolvimento da doença, enquanto a contribuição Africana aumentou proteção. No que diz respeito a análise diferencial do perfil de expressão dos microRNAs de pacientes com hanseníase, por meio da análise de biopsias de pele, revelaram-se 67 miRNAs diferencialmente expressos, dos quais 43 apresentavam um padrão de expressão downregulated e 24 upregulated. Quando analisamos amostras de sangue desses mesmos pacientes, observaram-se 10 miRNAs diferencialmente expressos, dos quais 9 com padrão de expressão downregulated e 1 upregulated. Os alvos pesquisados, em análise in silico, a partir desses resultados sugeriu os genes (IL1β, IL6, IL8, IL12, TLR2, TLR4, IL17RB, IFNGR1, TGFBR1, NFκβ, família SMAD, STAT3, CASP8, CYP19A1, BCL-2, entre outros) como envolvidos na patologia da hanseníase. Por fim, monstrou-se pela primeira vez o perfil de microRNAs em genome wide da Hanseníase. / Leprosy is caused by Mycobacterium leprae and patients can be grouped in Paucibacillary and Multibacillary. Alternatively, according by Ridley-Jopling (1966), using immune-hystogical criteria, grouped in two distinct pole: (i) Tuberculóide (TT); and (ii) lepromatous (LL), and your intermediaries. Independently these classification, the disease can be affected by molecules that modulates immune response, like genes that encode these molecules, and by small RNA (micro-RNA), wich regulated these genes, thus these study can improve the knowledge about the mechanism of response to infectious process, as well as enable the identification of new possibles biomarkers to assist diagnosis in leprosy. The objective of this study was to investigate eight INDEL polymorphisms on genes CYP19A1, NFKβ1, IL1α, CASP8, UGT1A1, PAR1, CYP2E1, and IL4, to identify possible susceptibility markers of leprosy and evaluate the influence of genetic ancestry on disease risk. Besides was performed the first genome wide miRNA profiling of Leprosy by next generation sequencing (NGS), assessing and describing the expression standard in leprosy. Our study shows that the NFKβ1, CASP8, PAR1, IL4 and CYP19A1 genes are possible markers for the susceptibility to development of leprosy and the severe clinical form MB. Moreover, after correcting for population structure within an admixture population, the results show that different levels of ethnic group composition can generate different OR rates for leprosy susceptibility. The differential expression profile from tissue samples reveal 67 miRNAs differentially expression, with 43 down and 24 upregulated and from blood sample were found a total of 10 miRNAs differentially expression with 9 down and one upregulated. Moreover was performed in silico target analysis and detect the genes (IL1β, IL6, IL8, IL12, TLR2, TLR4, IL17RB, IFNGR1, TGFBR1, NFκβ, família SMAD, STAT3, CASP8, CYP19A1, BCL-2, in others) involved on pathological of leprosy. Lastly, was showed for the first time the genome wide microRNA of leprosy.

O transtorno de déficit de atenção e hiperatividade (TDAH) é um dos transtornos psiquiátricos mais freqüentes da infância e adolescência, sendo caracterizado por sintomas de desatenção, hiperatividade e impulsividade. A contribuição genética na etiologia do TDAH é uma das mais altas já verificadas para transtornos psiquiátricos, com herdabilidade média estimada de 76%. Dentre os fatores genéticos que contribuiriam para o desenvolvimento da doença, genes que codificam componentes do sistema dopaminérgico estão entre os principais candidatos. Entre estes, o gene que codifica o receptor D4 de dopamina (DRD4) é o loco mais intensamente investigado nos estudos moleculares com o TDAH. O polimorfismo mais estudado no DRD4 é um VNTR de 48 pb localizado no exon 3; porém outros polimorfismos, localizados na região promotora do gene – uma duplicação de 120pb e os SNPs -521C>T e - 616C>G – também vêm sendo propostos como polimorfismos de suscetibilidade ao TDAH. Além desses, novas variantes em regiões regulatórias do gene, os SNPs rs11246227 e rs11246228, foram observados recentemente em associação com sintomas de desatenção do TDAH. O objetivo geral do presente trabalho foi aumentar a compreensão acerca da participação do gene DRD4 na etiologia do TDAH na nossa população Para tanto, foi testada inicialmente a possibilidade de associação do SNP rs11246227, sendo em seguida investigado o significado funcional dos SNPs rs11246227 e rs11246228, e sua possível relação com a doença, através de ferramentas de bioinformática. O estudo de associação foi realizado em uma amostra composta por 478 pacientes com TDAH, diagnosticados segundo os critérios do DSM-IV, e seus pais biológicos. O rs112466227 foi investigado por abordagens baseada em família (FBAT) e dimensional (PBAT, ANOVA). A possibilidade de desequilíbrio de ligação (DL) com polimorfismos previamente investigados na presente amostra foi estimada pelo programa MLocus. A análise in silico foi realizada utilizando diferentes bases de dados genômicos e programas de predição de sítios alvo para miRNAs e de funcionalidade. A análise pelo FBAT mostrou um desvio significativo da transmissão do alelo C nos pacientes do subtipo desatento. Foram observadas evidências de DL com a duplicação de 120bp e com o VNTR do exon 3. As análises de bioinformática mostraram que os SNPs rs11246227 e rs11246228 estão localizadas na região 3’ do gene DRD4, e não na região 5’, como previamente descrito. Diferenças entre os alelos, com perda ou ganho de sítios de ligação para diferentes miRNAs, foram detectados em ambos os SNPs pelos programas MicroInspector, 5 smiRNAdb e miRecords, e apenas no rs11246227 pelos programas Human miRNA Target e Mirò. A grande variabilidade e a complexidade genética marcante do gene DRD4 aliada à heterogeneidade fenotípica do TDAH provavelmente contribuíram para nossos resultados de associação, divergentes dos descritos na literatura, os quais necessitam de replicação em estudos futuros. Nossos achados em bioinformática sugerem um possível envolvimento dos SNPs investigados com a ligação de miRNAs relacionados aos processos de neurogênese e neuroplasticidade. Genes envolvidos com estes processos vêm sendo identificados nos genome-wide association studies realizados com o TDAH, o que apóia nossos resultados in silico. Entretanto, mais estudos funcionais são necessários, tanto in silico como in vitro, para esclarecer o envolvimento dos polimorfismos analisados na regulação da expressão do gene DRD4 via miRNAs e, consequentemente, do possível efeito desses elementos na etiologia da doença. / Attention-deficit/hyperactivity disorder (ADHD) is one of the most common psychiatric disorders of childhood and adolescence, characterized by inattentive, hyperactive and impulsive symptoms. Genetic contribution to ADHD etiology is one of the highest ever recorded for psychiatric disorders, with a mean heritability of 76%. Among genetic factors that could contribute to disorder development, genes encoding components from dopaminergic system are the main candidate. Of these, the dopamine D4 receptor gene (DRD4) is the most extensively investigated locus in molecular studies of ADHD. The most studied polymorphism in DRD4 gene is a variable number of tandem repeats (VNTR) of 48bp, located at exon 3, although other polymorphisms, located in promoter region – a 120bp duplication and the SNPs -521C> T and-616C> G – have also been proposed as susceptibility polymorphisms for ADHD. In addition, new variants in regulatory regions, the SNPs rs11246227 and rs11246228, have recently been associated with inattentive symptoms of the disorder. The overall objective of this study was to increase the understanding on the involvement of DRD4 gene in ADHD etiology in our population For this purpose, the possibility of association with the SNP rs11246227 was initially tested, being afterwards investigated the functional effect of both rs11246227 and rs11246228 and their possible relation to ADHD through bioinformatics approach. The association study was performed in a sample composed by 478 ADHD patients, diagnosed according to DSM-IV criteria, and their biological parents. The rs112466227 was investigated by both family-based (FBAT) and dimensional (PBAT, ANOVA) approaches. The possibility of linkage disequilibrium (LD) with polymorphisms previously investigated in the present sample was estimated by MLocus software. In silico analysis was conducted using different genomic databases and programs to predict miRNA target sites and functionality. FBAT analysis showed a significant excess of C allele transmission in inattentive subtype patients. Evidences of LD with both 120bp tandem duplication and exon 3 VNTR were observed. Bioinformatics analyses showed that both SNPs rs11246227 and rs11246228 are located in the 3' region of DRD4 gene, and not at 5’ region, as previously described. Differences between alleles, with loss or gain of binding sites, were detected in both SNPs by MicroInspector, smiRNAdb and miRecords, and only in rs11246227 by Human miRNA Targets and miRò DRD4 huge variability and marked genetic complexity allied to ADHD phenotypic heterogeneity might have contributed to our 7 association results, distinct from the ones reported in literature, what needs to be replicated in future studies. Our bioinformatics findings suggest a possible involvement of investigated SNPs in binding properties of miRNAs related to processes of neurogenesis and neuronal plasticity. Genes involved in these processes have been identified in ADHD genome-wide association studies, reinforcing our in silico results. However, new functional studies, using both in silico and in vitro approaches, are needed to clarify the involvement of the investigated polymorphisms in DRD4 expression control mediated by miRNAs and, consequently, the possible effect of these elements in ADHD etiology.

Os microRNAs são pequenos RNAs, não-codificantes que funcionam como reguladores a nível pós-transcricional da expressão gênica. Nos últimos anos, novas evidências demonstram o papel importante dos microRNAs na regulação e desenvolvimento do sistema imune. Apesar da função de poucos microRNAs ser conhecida, a sua expressão alterada vêm sendo associada a patogênese de diversas doenças autoimunes, incluindo a artrite reumatóide (AR). Recentemente a expressão desregulada de uma série de microRNAs está sendo descrita em pacientes com AR, e o papel patogênico de apenas uma parte deles foi investigada em modelos animais. A artrite reumatóide é uma doença autoimune sistêmica caracterizada por um intenso processo inflamatório na sinóvia, podendo causar destruição óssea e articular. Os linfócitos T apresentam papel importante na indução, manutenção e progressão da doença. A artrite induzida por colágeno é um modelo animal amplamente utilizado por suas características fisiopatológicas muito similares à doença em humanos. A linhagem de camundongos DBA-1/J desenvolve a doença após imunização e booster com colágeno do tipo II, enquanto que a linhagem DBA-2/J se mostra refratária. Isso confere um sistema modelo de susceptibilidade/resistência à artrite, que pode ser estudado em diferentes abordagens. O objetivo do nosso estudo foi identificar o perfil transcricional e as redes de interação entre um grupo de microRNAs e seus respectivos alvos nos timócitos e linfócitos T CD3+ periféricos nos camundongos da linhagem DBA-1/J e DBA-2/J. Para a avaliação da expressão gênica, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos entre as duas linhagens utilizadas. Novos microRNAs foram encontrados nos diferentes estágios de desenvolvimento do linfócito T. Nas redes de interação microRNA-RNAm obtidas, genes importantes associados aos processos de sistema imune, adesão e diferenciação celular, apoptose, recombinação, ativação de linfócitos T e resposta inflamatória, foram encontrados como potenciais alvos. Além disso, em uma perspectiva clínica, baseados nos resultados obtidos em camundongo, nos encontramos a expressão do miR-505 nos linfócitos T de pacientes com AR. Nossos resultados contribuem para a melhor compreensão dos mecanismos molecular envolvidos na resistência/susceptibilidade a CIA. / MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. During the last several years, evidence has emerged to show their critical role for the regulation and development of immune system. Although the function of most mammalian miRNAs has yet to be determined, their aberrant expression has been associated with several autoimmune conditions, including rheumatoid arthritis (RA). Recently, the deregulated expression of a dozen miRNAs has been reported in patients with RA, and the pathogenic role of only a few of these has been investigated in experimental mouse models. RA is a systemic autoimmune disorder mainly characterized by the inflammation of synovial tissue that can lead to destruction of bone and cartilage. The role of effectors T cells in induction, maintenance and progression of the disease is now becoming better understood. Collagen-induced arthritis is an animal model widely studied due to its similarities to human disease. The DBA-1/J mouse strain develops arthritis after immunization process and booster with Type II collagen, and the DBA-2/J strain is refractory to the disease induction. This offers an useful susceptibility/resistance model-system to study RA. The aim of this study was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes in DBA-1/J and DBA-2/J mice strain. For this purpose we used the microarray technology to evaluate the expression of the miRNAs and mRNAs as possible targets involved in this process. The use of bioinformatics software to reconstruct the networks was essential. The results show differential expression of mRNAs and miRNAs in thymocytes and peripheral CD3+ T lymphocytes between both strains. New miRNAs were found during all the stages of T cells development. The microRNA-mRNA interaction networks obtained in this study showed that important genes related to apoptose, immune system, recombination, cell adhesion and differentiation, inflammatory process and T cell activation were found as potential targets. In addition, in a clinical prospects, based on the results obtained in mice, we found the expression of the miRNA miR-505 in T cells of RA patients. Our results contribute to a better understand of the molecular mechanisms evolved in the resistance/susceptibility to CIA.

microRNAs are single-stranded small RNAs that function as post-transcriptional regulators of gene expression. We are studying the mir-44 family, specifically mir-44 and mir-45, which have identical sequence. Loss of mir-44 and mir-45 results in defects that suggest that the mir-44 family acts to negatively regulate the MAPK pathway. The MAPK pathway regulates sex myoblast migration, a process which is required for normal egg laying. We hypothesized that the mir-44 family of microRNAs is necessary for normal sex myoblast migration and subsequent formation of the functional egg laying structure in the hermaphrodite. We created a mutant that had mutations in both mir-44 and mir-45 and a transgene that expresses GFP in the sex myoblast cells. Then we observed the migration and division of the sex myoblasts in wild-type and mutant worms using fluorescence microscopy. In all cases, the mutant worms displayed a greater percent difference from average sex myoblast migration and division. However, a two-tailed two-proportions z-test found no significant difference between wild type and mutant sex myoblast migration (p=0.9148), nor in mutant sex myoblast division along the axial (p=0.4205) and sagittal (p=0.3583) planes of the body. This allows us to conclude that mir-44 and mir-45 are unlikely to be responsible for the migration nor division of the sex myoblasts, and the defects are likely due to interference with a different biological mechanism.

In the Kuehn lab, it has been shown that inducible pluripotent stems cells that have been induced to be trabecular meshwork cell-like (iPSC-TM) have a unique ability to regenerate dysfunctional trabecular meshwork (TM) cells by sharing specific unknown factors. In this thesis will discuss the novel means by which I isolate primary human Trabecular Meshwork (pTMs) and efficiently prepare cell cultures for experimentation, such as a sequencing experiment in which I studied expression changes that arose when the TM cell culture’s cell cycle control is manipulated. Previous research has shown that pTM grow atypical when 100% confluent compared to other epithelial cells creating an interesting time frame by which to observe their unique cell cycle control. Using newly isolated TM cell cultures I investigated expression of mRNA and miRNA to understand their roles in cell cycle control of these atypical cultures. With regards to the isolation of TM cell cultures were able to show that the “Crawling Out” methodology is an effective way to establish a pure TM cell line with both a low contamination rate and less passages/time. With these cultures we were able to establish 50 mRNAs and 19 miRNAs that were differential expressed in the TM cell cultures that were atypically grown. When reviewing the literature many of these expression changes were linked to carcinogenics, and the progression/prognosis of various cancer types.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

Im Rahmen des ersten Teils der vorliegenden Doktorarbeit konnten zwei nicht-essentielle (rps15, rpl36) und fünf essentielle (rps3, rps16, rpl22, rpl23, rpl32) im Plastom von Nicotiana tabacum kodierte Proteine des plastidären Ribosoms bezüglich ihrer Essentialität charakterisiert werden. Diese Gene wurden durch gezielte Knockout-Experimente inaktiviert und die resultierenden Effekte untersucht. Die Ergebnisse lassen einen Rückschluss auf die Lokalisation der Gene der insgesamt sieben untersuchten ribosomalen Proteine zu, die im Plastom mehrerer parasitischer, Plastiden-besitzender Spezies nicht mehr nachweisbar sind. Im Fall von rps15 könnte tatsächlich ein Verlust des Genes stattgefunden haben, im Fall der restlichen Gene ist eher mit einem Transfer in den Nukleus zu rechnen (rpl36 ausgenommen). Dies würde bedeuten, dass die Geschwindigkeit der erfolgreichen Etablierung eines Gentransfers in vielen parasitischen Spezies gegenüber grünen Pflanzen stark erhöht ist. Alle in E. coli nicht-essentiellen Proteine mit Homologen in Plastiden (rps15, rpl33, rpl36) sind auch dort, trotz ~1,5 Milliarden Jahren getrennter Evolution, nicht essentiell. Dieses Ergebnis bestätigt den schon früher festgestellten hohen Konservierungsgrad der bakteriellen und plastidären Translationsmaschinerien. Die Phänotypen der KO-Pflanzen der nicht-essentiellen Gene (rps15, rpl36) weisen auf eine interessante Rolle von S15 während der Ribosomenassemblierung hin und im Fall von L36 auf eine wichtige funktionelle Rolle im Plastiden-Ribosomen sowie auf eine Involvierung der Plastidentranslation in der Generierung eines retrograden Signals, welches die Blattform zu beeinflussen im Stande ist. Des Weiteren konnte eine Verbindung der Translationsaktivität mit der Ausbildung von Seitentrieben hergestellt werden, die vermutlich auf veränderte Auxinsynthese im Chloroplast zurückzuführen ist. Aus dem Folgeprojekt, bei dem Doppel-KO-Pflanzen nicht-essentieller ribosomaler Proteine erzeugt wurden, lässt sich auf eine relativ große Plastizität der Architektur von Plastidenribosomen schließen. Im zweiten Teil der Arbeit konnte erfolgreich ein Hochdurchsatz-Screeningsystem zur semiquantitativen Analyse von 192 verschiedenen miRNAs aus Chlamydomonas reinhardtii etabliert werden. Es gelang durch die Untersuchung von 23 verschiedenen Wachstums- und Stressbedingungen sowie Entwicklungsstadien mehrere miRNAs zu identifizieren, die eine differenzielle Expression zeigen sowie unter allen untersuchten Bedingungen konstant bleibende miRNAs nachzuweisen. Dadurch konnten mehrere vielversprechende Kandidaten-miRNAs ausgemacht werden, die nun eingehender untersucht werden können. / Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway. In the second project a qRT-PCR based plattform for the analysis of miRNAs in Chlamydomonas reinhardtii has been developed. 20 different growth conditions have been scanned.