@phdthesis{Tyrsin2008,
  author    = {Tyrsin, Dmitry},
  title     = {Autoregulation of NFATc1 gene},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-26544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Die Familie der NFAT-Transkriptionsfaktoren (NFATc1-c4) ist im Zuge einer Immunreaktion endscheidend an der transkriptionellen Regulation der Genexpression beteiligt. Wurden NFAT-Faktoren zun{\"a}chst als T-zell-spezifische Aktivatoren von Zytokinpromotoren beschrieben, so hat sich inzwischen gezeigt, dass sie in einer Vielzahl von Geweben eine wichtige Rolle spielen. Als Beispiele seien die Herzklappenentwicklung, die Bildung von Blutgef{\"a}ssen, die Ausbildung neuronaler Axone oder die Osteoklastendifferenzierung genannt [10, 24]. In der hier vorliegenden Arbeit zeigen wir, dass die starke Expression der kurzen Isoform NFATc1/\&\#945;A in Effektor-T-Lymphozyten durch die induzierbare Aktivit{\"a}t des Promoters P1 kontrolliert wird. Die P1 Aktivierung f{\"u}hrt zum Splicing des Exon 1 zu 3 (\&\#945;-Isoformen) und endet meist durch Benutzung der Polyadenylierungsstelle pA1 hinter Exon 9 (A-Isoformen). Der zweite, schw{\"a}cherer Promoter P2 befindet sich vor dem zweiten Exon und ist f{\"u}r die konstitutive Synthese der \&\#946;-Isoformen verantwortlich. Der Transkriptionstart am zweiten Exon geht meist mit der Benutzung einer zweiten, hinter dem 11. Exon gelegenen Polyadenylierungsstelle pA2 einher, die durch alternatives Splicing zur Synthese der Isoformen B und C f{\"u}hrt. Insgesamt k{\"o}nnen so vom nfatc1-Lokus sechs verschiedene Isoformen (\&\#945;A, \&\#945;B, \&\#945;C, \&\#946;A, \&\#946;B und \&\#946;C) generiert werden. Die induzierbare Aktivit{\"a}t des P1-Promoters ist, im Gegensatz zum eher konstitutiv aktiven P2-Promoter, NFAT-abh{\"a}ngig und somit eine Form der Autoregulation. In ruhenden T-Lymphozyten sind einzig die Transkripte der NFATc1/\&\#946;-Isoformen nachweisbar. Nach einer T-Zell-Aktivierung nimmt ihre H{\"a}ufigkeit dann ab, w{\"a}hrend nun die \&\#945;-Isoformen dominant werden. In dieser Arbeit wird gezeigt, dass es nach Induktion prim{\"a}rer Effektor-T-Helfer-Zellen oder in T-Zell-Linien zu einer 15-20-fachen Akkumulation der NFATc1/\&\#945;A mRNA bzw. einer 2-5-fachen Zunahme der NFATc1/\&\#945;B und C mRNAs kommt. Zur maximalen Induktion des P1-Promotors bedarf es zum einen eines anhaltenden Anstiegs der intrazellul{\"a}ren Kalziumkonzentration, die zur Aktivierung der Phosphatase Calcineurin und damit zur Kernlokalisation der NFAT-Faktoren f{\"u}hrt. Zum anderen ist die Aktivierung der Proteinkinase C-Enzyme und der MAP-Kinasen notwendig, wie sie durch Phorbolester in der Zelle vermittelt wird. Dies l{\"a}sst darauf schließen, dass f{\"u}r eine optimale Aktivierung des P1-Promotors sowohl Signale des T-Zell-Rezeptors als auch Signale von Korezeptoren - wie von CD28 - notwendig sind. Da die Induktion von NFATc1/\&\#945;A in NFATc2/NFATc3 doppeldefizienten M{\"a}usen normal erfolgt, kann man schlussfolgern, dass NFATc1 in Form einer Autoregulation die Aktivit{\"a}t des P1-Promoters und damit die Synthese der \&\#945;-Isoformen kontrolliert. Die NFAT-vermittelte Aktivierung des P1-Promoters erfolgt {\"u}ber zwei tandemartig angeordnete NFAT-Bindungsstellen der Nukleotidsequenz TGGAAA, an die jeweils ein NFAT-Protein binden kann. Daneben enth{\"a}lt der Promoter konservierte Bindemotive f{\"u}r CREB-, AP-1, Sp-, NF-kB- und GATA-Faktoren, die wahrscheinlich an der komplexen Kontrolle dieses induzierbaren NFATc1-Promoters beteiligt sind. Zusammengefasst ergibt sich aus diesen Daten das folgende Modell. Die Transkription im nfatc1-Genlokus erfolgt in naiven und in ruhenden Effektor-T-Zellen konstitutiv und gesteuert durch den P2-Promotor. In Folge einer Aktivierung der Zelle verringert sich die Aktivit{\"a}t des P2-Promotors, w{\"a}hrend gleichzeitig der P1-Promotor induziert wird, der zusammen mit einer verst{\"a}rkten Nutzung der pA1-Polyadenylierungssequenz f{\"u}r die massive Zunahme der NFATc1/\&\#945;A-Isoform verantwortlich ist. Dies deutet auf eine besondere Bedeutung dieser kurzen Isoform in der Effektorphase der T-Zell-Aktivierung hin, insbesondere in Th1-Zellen, die NFATc1/\&\#945;A in hohen Konzentrationen produzieren.},
  subject      = {Autoregulation},
  language  = {en}
}
@phdthesis{Dugar2016,
  author    = {Dugar, Gaurav},
  title     = {Comparative transcriptomics and post-transcriptional regulation in \(Campylobacter\) \(jejuni\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146180},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The transcriptome is defined as the set of all RNA molecules transcribed in a cell. These include protein-coding messenger RNAs (mRNAs) as well as non-coding RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small non-coding RNAs (sRNAs). sRNAs are known to play an important role in regulating gene expression and virulence in pathogens. In this thesis, the transcriptome of the food-borne pathogen Campylobacter jejuni was characterized at single nucleotide resolution by use of next-generation sequencing approaches. The first genome of a C. jejuni strain was published in the year 2000. However, its transcriptome remained uncharacterized at large. C. jejuni can survive in a variety of ecological niches and hosts. However, how strain-specific transcriptional changes contribute to such adaptation is not known. In this study, the global transcriptome maps of four closely related C. jejuni strains were defined using a differential RNA-seq (dRNA-seq) approach. This analysis also included a novel automated method to annotate the transcriptional start sites (TSS) at a genome-wide scale. Next, the transcriptomes of four strains were simultaneously mapped and compared by the use of a common coordinate system derived from whole-genome alignment, termed as SuperGenome. This approach helped to refine the promoter maps by comparison of TSS within strains. Most of the TSS were found to be conserved among all four strains, but some single-nucleotide-polymorphisms (SNPs) around promoter regions led to strain-specific transcriptional output. Most of these SNPs altered transcription only slightly, but some others led to a complete abrogation of transcription leading to differential molecular phenotypes. These in turn might help the strains to adapt to their specific host or microniche. The transcriptome also unveiled a plethora of sRNAs, some of which were conserved among the four strains while others were strain specific. Furthermore, a Cas9-dependent minimal type-II CRISPR-Cas system with only three Cas genes and multiple promoters to drive the transcription of the CRISPR locus was also characterized in C. jejuni using the dRNA-seq dataset. Apart from sRNAs, the role of global RNA binding proteins (RBPs) is also unclear in C. jejuni. Aided by the global transcriptome data, the role of RBPs in post-transcriptional regulation of C. jejuni was studied at a global scale. Two of the most widely studied RNA binding proteins in bacteria are Hfq and CsrA. The RNA interactome of the translational regulator CsrA was defined using another global deep-sequencing technique that combines co-immunoprecipitation (coIP) with RNA sequencing (RIP-seq). Using this interactome dataset, the direct targets of this widespread global post-transcriptional regulator were defined, revealing a significant enrichment for mRNAs encoding genes involved in flagella biosynthesis. Unlike Gammaproteobacteria, where sRNAs such as CsrB/C, antagonize CsrA activity, no sRNAs were enriched in the CsrA-coIP in C. jejuni, indicating absence of any sRNA antagonists and novel modes of CsrA activity regulation. Instead, the CsrA regulatory pathway revealed flaA mRNA, encoding the major flagellin, as a dual-function mRNA. flaA mRNA was the main target of CsrA but it also served to antagonize CsrA activity along with the protein antagonist FliW previously identified in the Gram-positive bacterium Bacillus subtilis. Furthermore, this regulatory mRNA was also shown in this thesis to localize to the poles of elongating C. jejuni cells in a translation-dependent manner. It was also shown that this localization is dependent on the CsrA-FliW regulon, which controls the translation of flaA mRNA. The role and mechanism of flaA mRNA localization or mRNA localization in general is not yet clear in bacteria when compared to their eukaryotic counterparts. Overall, this study provides first insights into riboregulation of the bacterial pathogen C. jejuni. The work presented in this thesis unveils several novel modes of riboregulation in C. jejuni, which could be applicable more generally. Moreover, this study also lays out several unsolved intriguing questions, which may pave the way for interesting studies to come.},
  subject      = {Campylobacter jejuni},
  language  = {en}
}
@phdthesis{VasquezOspina2016,
  author    = {Vasquez Ospina, Juan Jose},
  title     = {Development of tools for the study of gene regulation in Trypanosoma brucei},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133996},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The protozoan parasite Trypanosoma brucei is the causal agent of sleeping sickness and besides its epidemiological importance it has been used as model organism for the study of many aspects of cellular and molecular biology especially the post-transcriptional control of gene expression. Several studies in the last 30 years have shown the importance of mRNA processing and stability for gene regulation. In T. brucei genes are unusually arranged in polycistronic transcription units (PTUs) and a coupled process of trans-splicing and polyadenylation produces the mature mRNAs. Both processes, mRNA processing and stability, cannot completely explain the control of gene expression in the different life cycle stages analyzed in T. brucei so far. In recent years, the relevance of expression regulation at the level of translation has become evident in other eukaryotes. Therefore, in the first part of my thesis I studied the impact of translational regulation by means of a genome-wide ribosome profiling approach. My data suggest that translational efficiencies vary between life cycle stages of the parasite as well as between genes within one life cycle stage. Furthermore, using ribosome profiling I was able to identify many new putative un-annotated coding sequences and to evaluate the coding potential of upstream open reading frames (uORF). Comparing my results with previously published proteomic and RNA interference (RNAi) target sequencing (RIT-seq) datasets allowed me to validate some of the new coding sequences and to evaluate their relevance for the fitness of the parasite. In the second part of my thesis I used the transcriptomic and translatomic profiles obtained from the ribosome profiling analysis for the identification of putative non-coding RNAs (ncRNAs). These results led to the analysis of the coding potential in the regions upstream and downstream of the expressed variant surface glycoprotein (VSG), which is outlined in the third part of the results section. The region upstream of the VSG, the co-transposed region (CTR), has been implicated in an increase of the in situ switching rate upon its deletion. The ribosome profiling results indicated moderate transcription but not translation in this region. These results raised the possibility that the CTR may be transcribed into ncRNA. Therefore, in the third part of my thesis, I performed a primary characterization of the CTR-derived transcripts based on northern blotting and RACE. The results suggested the presence of a unique transcript species of about 1,200 nucleotides (nt) and polyadenylated at the 3'-end of the sequence. The deletion of the CTR sequence promoting and increase of the in situ switching rates was performed around 20 years ago by means of inserting reporter genes. With the recent development of endonuclease-based tools for genome editing, it is now possible to delete sequences in a marker-free way. In the fourth part of my thesis, I show the results on the implementation of the highly efficient genome-editing CRISPR-Cas9 system in T. brucei using episomes. As a proof of principle, I inserted the sequence coding for the enhanced green fluorescent protein (eGFP) at the end of the SCD6 coding sequence (CDS). Fluorescent cells were observed as early as two days after transfection. Therefore, after the successful set up of the CRISPR-Cas9 system it will be possible to modify genomic regions with more relevance for the biology of the parasite, such as the substitution of codons present in gene tandem arrays. The implementation of ribosome profiling in T. brucei opens the opportunity for the study of translational regulation in a genome-wide scale, the re-annotation of the currently available genome, the search for new putative coding sequences, the detection of putative ncRNAs, the evaluation of the coding potential in uORFs and the role of unstranslated regions (UTRs) in the regulation of translation. In turn, the implementation of the CRISPR-Cas9 system offers the possibility to manipulate the genome of the parasite at a nucleotide resolution and without the need of including resistant makers. The CRISPR-Cas9 system is a powerful tool for editing ncRNAs, UTRs, multicopy gene families and CDSs keeping their endogenous UTRs. Moreover, the system can be used for the modification of both alleles after just one round of transfection and of codons coding for amino acids carrying post-translational modifications (PTMs) among other possibilities.    },
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{Kern2014,
  author    = {Kern, Selina Melanie},
  title     = {Functional characterization of splicing-associated kinases in the blood stages of the malaria parasite Plasmodium falciparum},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115219},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Besides HIV and tuberculosis, malaria still is one of the most devastating infectious diseases especially in developing countries, with Plasmodium falciparum being responsible for the frequently lethal form of malaria tropica. It is a major cause of mortality as well as morbidity, whereby pregnant women and children under the age of five years are most severely affected. Rapidly emerging drug resistances and the lack of an effective and safe vaccine hamper the combat against malaria by chemical and pharmacological regimens, and moreover the poor socio-economic and healthcare conditions in malaria-endemic countries are compromising the extermination of this deadly tropical disease to a large extent. Malaria research is still questing for druggable targets in the parasitic protozoan which pledge to be refractory against evolving resistance-mediating mutations and yet constitute affordable and compliant antimalarial chemotherapeutics. The parasite kinome consists of members that represent most eukaryotic protein kinase groups, but also contains several groups that can not be assigned to conservative ePK groups. Moreover, given the remarkable divergence of plasmodial kinases in respect to the human host kinome and the fact that several plasmodial kinases have been identified that are essential for the intraerythrocytic developmental cycle, these parasite enzymes represent auspicious targets for antimalarial regimens. Despite elaborate investigations on several other ePK groups, merely scant research has been conducted regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family, PfCLK-1-4. In other eukaryotes, CLKs are involved in mRNA processing and splicing by means of phosphorylation of serine/arginine-rich (SR) proteins, which are crucial components of the splicing machinery in the alternative splicing pathway. All four PfCLKs are abundantly expressed in asexual parasites and gametocytes, and stage-specific expression profiles of PfCLK-1 and PfCLK-2 exhibited nucleus-associated localization and an association with phosphorylation activity. In the course of this study, PfCLK-3 and PfCLK-4 were functionally characterized by indirect immunofluorescence, Western blot analysis and kinase activity assays. These data confirm that the two kinases are primarily expressed in the nucleus of trophozoites and both kinases possess in vitro phosphorylation activity on physiological substrates. Likewise PfCLK-1 and PfCLK-2, reverse genetic studies exhibited the indispensability of both PfCLKs on the asexual life cycle of P. falciparum, rendering them as potential candidates for antiplasmodial strategies. Moreover, this study was conducted to identify putative SR proteins as substrates of all four PfCLKs. Previous alignments revealed a significant homology of the parasite CLKs to yeast SR protein kinase Sky1p. Kinase activity assays showed in vitro phosphorylation of the yeast Sky1p substrate and SR protein Npl3p by precipitated PfCLKs. In addition, four homologous plasmodial SR proteins were identified that are phosphorylated by PfCLKs in vitro: PfASF-1, PFSRSF12, PfSFRS4 and PfSR-1. All four parasite SR splicing factors are predominantly expressed in the nuclei of trophozoites. For PfCLK-1, a co-localization with the SR proteins was verified. Finally, a library of human and microbial CLK inhibitors and the antiseptic chlorhexidine (CHX) was screened to determine their inhibitory effect on different parasite life cycle stages and on the PfCLKs specifically. Five inhibitors out of 63 compounds from the investigated library were selected that show a moderate inhibition on asexual life cycle stages with IC50 values ranging between approximately 4 and 8 µM. Noteworthy, these inhibitors belong to the substance classes of aminopyrimidines or oxo-β-carbolines. Actually, the antibiotic compound CHX demonstrated an IC50 in the low nanomolar range. Stage-of-inhibition assays revealed that CHX severely affects the formation of schizonts. All of the selected CLKs inhibitors also affect gametocytogenesis as well as gametogenesis, as scrutinized in gametocyte toxicity assays and exflagellation assays, respectively. Kinase activity assays confirm a specific inhibition of CLK-mediated phosphorylation of all four kinases, when the CLK inhibitors are applied on immunoprecipitated PfCLKs. These findings on PfCLK-inhibiting compounds are initial attempts to determine putative antimalarial compounds targeting the PfCLKs. Moreover, these results provide an effective means to generate chemical kinase KOs in order to phenotypically study the role of the PfCLKs especially in splicing events and mRNA metabolism. This approach of functionally characterizing the CLKs in P. falciparum is of particular interest since the malarial spliceosome is still poorly understood and will gain further insight into the parasite splicing machinery.},
  subject      = {Plasmodium falciparum},
  language  = {en}
}
@phdthesis{Bergmann2011,
  author    = {Bergmann, Anna},
  title     = {Untersuchungen zur Verwertung proteinhaltiger Substrate als m{\"o}gliche Virulenzdeterminante des humanpathogenen Schimmelpilzes Aspergillus fumigatus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67333},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Die asexuellen Sporen von Aspergillus fumigatus sind ubiquit{\"a}r verbreitete Luftkeime. Als Saprophyt ist dieser opportunistisch humanpathogene Pilz darauf spezialisiert, polymere Substanzen aus dem umgebenden Milieu zu zersetzen, um daraus die von ihm ben{\"o}tigten N{\"a}hrstoffe zu generieren und aufzunehmen. Die F{\"a}higkeit, verschiedene Stickstoff- und Kohlenstoffquellen zu verwerten, tr{\"a}gt dabei zu seiner Virulenz bei und hierbei scheint die extrazellul{\"a}re Proteolyse eine wichtige Rolle zu spielen. Sekretierte Proteasen, die das umgebende Gewebe w{\"a}hrend einer Infektion mit A. fumigatus erschließen, k{\"o}nnten somit zu dessen Pathogenit{\"a}t beitragen. Dementsprechend sollte im Rahmen dieser Arbeit die Bedeutung einer Regulation der extrazellul{\"a}ren proteolytischen Aktivit{\"a}t von A. fumigatus f{\"u}r dessen Virulenz untersucht werden. Dies geschah durch Untersuchungen eines konservierten Transkriptionsfaktors, PrtT. Dabei stellte sich heraus, dass PrtT die Expression der drei Hauptproteasen von A. fumigatus, Alp, Mep und Pep stark beeinflusst, in einem murinen Tiermodell der pulmonaren Aspergillose scheint dieser Regulator jedoch keine Rolle f{\"u}r die Pathogenit{\"a}t von A. fumigatus zu spielen. Um einen weiteren Aspekt des pilzlichen Aminos{\"a}urestoffwechsels zu beleuchten, wurde die Biosynthese der aromatischen Aminos{\"a}uren als m{\"o}gliche Virulenzdeterminate untersucht. F{\"u}r den Menschen sind diese Aminos{\"a}uren essentiell, weshalb dieser Syntheseweg ein m{\"o}gliches Ziel f{\"u}r antimykotische Substanzen darstellen k{\"o}nnte. Es konnten mehrere f{\"u}r A. fumigatus essentielle Komponenten des Shikimatweges identifiziert werden, des Weiteren wurden Deletionsmutanten in den Genen aroC und trpA, die f{\"u}r die Chorismatmutase bzw. Anthranilatsynthase der Biosynthese von Phenylalanin und Tyrosin bzw. Tryptophan kodieren, erzeugt und ph{\"a}notypisch charakterisiert. Deren Untersuchung in einem alternativen Tiermodell der Aspergillose zeigte eine deutlich attenuierte Virulenz. Diese Ergebnisse verdeutlichen, wie wichtig die Biosynthese der aromatischen Aminos{\"a}uren f{\"u}r das Wachstum von A. fumigatus ist, und dass ein Eingriff in diesen Syntheseweg eine lohnende Strategie zur Entwicklung neuer Antimykotika sein k{\"o}nnte. Die hier pr{\"a}sentierten Ergebnisse unterstreichen die f{\"u}r den Schimmelpilz A. fumigatus typische Redundanz bez{\"u}glich extrazellul{\"a}rer proteolytischer Enzyme und dass diese nur bedingt hinsichtlich ihres Virulenzbeitrags untersucht werden k{\"o}nnen. Im Gegensatz hierzu lassen sich bestimmte Stoffwechselwege, die oftmals durch einzigartige Genprodukte katalysiert werden, unter Umst{\"a}nden besser als unspezifische aber vielversprechende Virulenzdeterminanten identifizieren.},
  subject      = {Aspergillus fumigatus},
  language  = {de}
}