Invasive Zygomykosen verzeichnen in den letzten Jahren eine steigende Inzidenz, insbesondere im Risikokollektiv immunsupprimierter Patienten. Aufgrund des häufig letalen Verlaufs dieser Infektionen ist eine rasche, korrekte Diagnosestellung essentiell, um rechtzeitig eine adäquate Therapie einzuleiten. Jedoch sieht sich die konventionelle, mikrobiologische Diagnostik mit vielen Problemen konfrontiert, so dass molekularbiologische Nachweisverfahren zunehmend in den Fokus der Aufmerksamkeit rücken. Eine zuverlässige, mit relativ geringem Zeit- und Kostenaufwand praktizierbare Methode stellt in diesem Zusammenhang die Real-time-PCR dar, deren Aussagekraft durch anschließende Speziesidentifizierung mittels Sequenzierung noch verstärkt werden kann. Aus diesem Grund wurden im Rahmen dieser Arbeit 3 PCR-Assays entwickelt und deren Sensitivität, Spezifität und klinische Anwendbarkeit evaluiert. Alle 3 Systeme nutzten Multi-copy-Gene des ribosomalen Operons der Zygomyzeten als Target und erwiesen sich als zuverlässige Werkzeuge zur Amplifikation fungaler DNA. Sie wurde sowohl an Pilzkulturen, als auch an klinischen Proben und einem Quasi-Tiermodell mit Erfolg ausgetestet und werden möglicherweise in Zukunft der klinischen Routinediagnostik zur Verfügung stehen. Bedingt durch die Seltenheit invasiver Zygomykosen besteht in diesem Bereich noch ein großer Forschungsbedarf, auch, um die noch nicht optimale Therapie dieser Erkrankungen zu verbessern. Es bleibt daher zu hoffen, dass sich in absehbarer Zeit mehr Forschungsgruppen mit diesen Erregern beschäftigen, damit den schwer kranken Patienten eine echte Heilungschance geboten werden kann. / During the last years invasive zygomycosis register a rising incidence, in particular in the risk collective of immunosuppressed patients. On account of the often lethal course of these infections a fast, correct diagnosis is essential to initiate an adequate therapy on time. However, the conventional, microbiological diagnostics is confronted with many problems, so that molecular-biological methods to detect invasive mucormycosis get moor important. A reliable method requiring relatively low time is the real-time-PCR. The following sequencing of the amplicon allows the identification to the genus level. That's why 3 PCR-Assays were developed and their sensitivity, specificity and clinical applicability were evaluated. All 3 systems targeted multi copy genes of the ribosomal operon of the zygomycetes and turned out to be reliable tools for the amplification of fungal DNA. They were successfully tested in fungal cultures, as well as in clinical tests and a quasi animal model. In future they will possibly be available to the clinical routine diagnostics. Because of the rarity of invasive zycomycosis a lot of research is needed in this area, also to improve the therapy, frequently being insufficient. However, there remains hope that in future more research groups deal with these causes, so that a real healing chance can be offered to the seriously ill patients.

Most microbial organic molecules are secondary metabolites which consist of diverse chemical structures and a range of biological activities. Actinobacteria form a large group of Eubacteria that are prolific producers of these metabolites. The recurrence of pathogens resistant to antibiotics and a wider use of these metabolites apart from their use as anti-infectives, has been the impetus for pharmaceutical companies to search for compounds produced by rare and existing actinobacterial cultures. Accessing microbial biosynthetic pathway diversity has been possible through the use of sensitive and innovative molecular detection methodologies. The present study evaluated the use of molecular based screening as a rational approach to detect secondary metabolite biosynthetic genes (SMBG) in uncharacterised natural Actinobacterial populations. A polymerase chain reaction (PCR) approach was selected for ease of application and high sample processivity. Rational designed screening approaches using PCR in the discovery of SMBG, involved identifying common functions in secondary metabolite biosynthetic pathways, such as condensation reactions in polyketide synthesis, genes encoding these functions, and using conserved regions of these genes as templates for the design of primers to detect similar sequences in uncharacterised actinobacteria. Design of primers involved rigorous in silico analysis followed by experimentation and validation. PCR screening was applied to 22 uncharacterised environmental isolates, eight of these displayed the presence of the ketosynthase (KS) gene belonging to the type I polyketide synthases and eight contained the ketosynthase (KSĄ) gene belonging to the type II polyketide synthases, six of the isolates contained the presence of a presumptive dTDP-glucose synthase (strD) gene which is involved in the formation of deoxysugar components of aminoglycoside antibiotics and one isolate contained the presence of a presumptive isopenicillin N synthase (pcbC) gene involved in beta-lactam synthesis. Alignments of partially sequenced PCR products from isolates A1488 and A3023 obtained using type II PKS primers showed close similarities with KSĄ genes from antibiotic producing actinobacteria. Similarly, alignments of sequences from isolates A1113 and A0350 showed regions of similarities to KS genes from antibiotic producing actinobacteria. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening it was determined that most of the isolates possessed the capacity to produce antimicrobial metabolites. Dominant antagonistic activity was detected against Gram positive bacteria and to a minor extent against fungi. Optimal fermentation liquid media were identified for certain isolates for the production of antimicrobial metabolites. Two alternative fermentation methods; solid-state and liquid-oil fermentations were evaluated to improve secondary metabolite production in the uncharacterised isolates. Solid-substrate fermentation showed that it could induce a complex metabolite pattern by TLC analysis, however this pattern varied according to the substrate being used. Liquid media supplemented with refined oils, showed a positive response indicated by higher antibacterial activities detected. Evaluation of semi-purified organic extracts identified two isolates A1113 and A0350 producing similar antimicrobial metabolites as detected by HPLC/UV/MS, a literature database search of similar compounds containing the same molecular weight identified the compound as belonging to the actinomycin group of compounds. A complex metabolic pattern was identified for isolate A2381, database searching identified some of the compounds as having similar molecular weights to actinopyrones, trichostatins, antibiotics PI 220, WP 3688-5 and YL 01869P. Drug discovery screening can serve to benefit from PCR detection of biochemical genotypes in initial screens, providing a rapid approach in identifying secondary metabolite producing capabilities of microorganisms prior to the commencement of costly and time consuming fermentation studies. Additionally the identification of biochemical genotypes allows a directed approach in using fermentation media designed to induce biosynthetic pathways of specific classes of compounds.

Several strain typing techniques are available to categorize Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) isolates into cattle, sheep, bison, and Intermediate groups. The majority of isolates studied were identified as members of the cattle associated group, regardless of sample host origin, suggesting that the cattle group of M. paratuberculosis isolates are very successful. This may be because host specificity is not critical for this group or the small differences required to demonstrate host specificity have not yet been found. A major limitation to the epidemiological study of M. paratuberculosis has been the difficulty associated with laboratory cultivation of this micro-organism. The new typing techniques described in this thesis do not require viable M. paratuberculosis bacteria and therefore open a door to novel typing practices. The new molecular techniques, single stranded conformation polymorphism (SSCP) analysis and satellite typing, were applied to M. paratuberculosis isolates (n=75) from a broad range of ruminant hosts and geographic locations. SSCP analysis and satellite typing were compared to currently accepted techniques (PCR-REA, RFLP, PFGE) for their ability to rapidly and reliably differentiate among M. paratuberculosis isolates. PCR-REA segregated isolates (n=75) into cattle (n=72), sheep (n=1) or bison (n=2) associated strain types. Two isolates from cattle in Canada were typed as RFLP-BstEII C5 by RFLP analysis. PFGE grouped a subset (n=8) of M. paratuberculosis isolates into 4 different PFGE types. Satellite typing resulted in 4 different satellite types (A, B, C, D). SSCP analysis identified 2 regions (IS900-2 and HSP70) where sequence polymorphisms could be targeted to display differences among M. paratuberculosis isolates.

En la última década del siglo XX, el campo de los microsistemas para análisis total (µ-TAS) y, más concretamente, el de los DNA-chips ha adquirido una importancia preponderante en el ámbito de los microsistemas. En gran parte, el creciente interés por estos dispositivos se debe a las substanciales mejoras que prometen: análisis más rápidos, baratos y automatizados, pero también es debido a la posibilidad de implementar técnicas analíticas antes impensables (e.g. chips de hibridación). En el caso particular de los DNA-chips, se han desarrollado prototipos funcionales para PCR, LCR, electroforesis en gel, di-electroforesis, hibridación y varias combinaciones de estas técnicas, al tiempo que los chips de hibridación masiva (mayoritariamente basados en arrayers) han llegado a convertirse en un éxito comercial. Aun así, y aunque se ha llevado a cabo mucho trabajo en estos años, es necesaria todavía mucha investigación para afrontar algunos de los principales retos de los DNA-chips. En el transcurso de esta tesis doctoral, se ha llevado a cabo el desarrollo un proceso tecnológico común para la fabricación de DNA-chips multifunción (i.e. sistemas versátiles basados en PCR y electroforesis), poniendo un especial énfasis en la compatibilidad con los procesos CMOS estándar, a fin de conseguir desarrollar prototipos proto-industriales. Como demostrador de esta puesta a punto tecnológica, se han diseñado, fabricado y testado chips de PCR, y la PCR en chips ha sido optimizada con respecto a materiales de fabricación, metodologías de inserción/extracción, composición bioquímica de la mix de PCR, diferentes configuraciones de calentadores/sensores (Peltier/termopares vs. resistencias integradas) y la cinética de la reacción. / In the last decade of the twentieth century, the fields of µ-TAS and, more specifically, DNA-chips have acquired increasing importance in the microsystems arena. The main reason for this surge of interest lies in the advantages these new devices seek to bring forth: faster, cheaper and completely automated analyses, and also in the outbreak of novel analytical techniques (e.g. hybridization chips). In the particular case of DNA-chips, functional prototypes have been demonstrated for PCR, LCR, gel electrophoresis, di-electrophoresis, hybridization and various combinations of these techniques, whilst hybridization chips (mainly arrayer chips) have become a successful market application. But, even though a considerable amount of work has been carried out in these few years, much research is still required to address fundamental problems of DNA-chips. In this doctoral work, a common-ground technological setup for the production of multifunction DNA-chips (i.e. PCR plus electrophoresis systems) has been laid down, placing strong emphasis in its compatibility with standard CMOS processes in order to produce proto-industrial prototypes. As a demonstrator of this technological setup, PCR-chips have been designed, manufactured and tested, and the chip PCR reaction has been optimized with respect to surface materials, insertion and extraction methods, biochemical mix composition, heater/sensor setups (Peltier/thermocouple vs. thin-film driven systems) and reaction kinetics.

Several strain typing techniques are available to categorize Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) isolates into cattle, sheep, bison, and Intermediate groups. The majority of isolates studied were identified as members of the cattle associated group, regardless of sample host origin, suggesting that the cattle group of M. paratuberculosis isolates are very successful. This may be because host specificity is not critical for this group or the small differences required to demonstrate host specificity have not yet been found. A major limitation to the epidemiological study of M. paratuberculosis has been the difficulty associated with laboratory cultivation of this micro-organism. The new typing techniques described in this thesis do not require viable M. paratuberculosis bacteria and therefore open a door to novel typing practices. The new molecular techniques, single stranded conformation polymorphism (SSCP) analysis and satellite typing, were applied to M. paratuberculosis isolates (n=75) from a broad range of ruminant hosts and geographic locations. SSCP analysis and satellite typing were compared to currently accepted techniques (PCR-REA, RFLP, PFGE) for their ability to rapidly and reliably differentiate among M. paratuberculosis isolates. PCR-REA segregated isolates (n=75) into cattle (n=72), sheep (n=1) or bison (n=2) associated strain types. Two isolates from cattle in Canada were typed as RFLP-BstEII C5 by RFLP analysis. PFGE grouped a subset (n=8) of M. paratuberculosis isolates into 4 different PFGE types. Satellite typing resulted in 4 different satellite types (A, B, C, D). SSCP analysis identified 2 regions (IS900-2 and HSP70) where sequence polymorphisms could be targeted to display differences among M. paratuberculosis isolates.

Flavonoid 3¡¦,5¡¦-hydroxylase (F3¡¦,5¡¦H) is the key enzyme that catalyzesthe anthocyanin biosynthesis pathway for the expression of blue or purple flower color. The garden crops of Verbena x hybrida were used to clone its F3¡¦,5¡¦H gene for the investigation of flower color engineering and regulatory program. Degenerate primers were designed from the conservative regions of other published F3¡¦,5¡¦H genes to amplify the expectant DNA fragment . A full-length cDNA of the F3¡¦,5¡¦H gene designated VhFH1 (AY604727) was cloned by the method of 5¡¦and 3¡¦ RACE, and its genomic DNA sequence was isolated by the IPCR strategy. Nucleotide sequence alignment revealed that VhFH1 contains two introns and a 1542 bp open reading frame encoding a polypeptide of 514 amino acid residues, and there could be a promoter sequence with TATA box signal in the upstream of the transcription start site. The amino acid sequence of VhFH1 was compared with the previous reported F3¡¦,5¡¦H and showed between 50¢Mand 77¢Midentity with those species. The expectant molecular mass and isoelectric point of VhFH1 protein is 57 KD and 7.69, respectively. There are three typical motifs of the F3¡¦,5¡¦H that belongs to the cytochrome P450 proteins in the VhFH1 predicted protein. According to the above-mentioned conjecture, VhFH1 is a full-length cDNA of the F3¡¦,5¡¦H gene in the V. hybrida. This cDNA fragment was inserted into the plant expression vector pCambia 1304 and could be detected the expression in E. coli by RT-PCR and protein electrophoresis. It is practicable to transform the horticultural plants with these vectors to create novel flower colors in the future. Furthermore, transcripts of the F3¡¦,5¡¦H gene were detected in the blue, purple and white flowers but not in the red one as revealed by RT-PCR. These results are advantageous in the further investigation of regulatory factors of the anthocyanin biosynthesis pathway in the V. hybrida.

Multiple polymerase chain reaction (multiple PCR) is one of the most important techniques in molecular biology. The selection of a suitable set of primers is very important for multiple PCR experiments. The primer selection problem is to minimize the number of primers required to amplify a set of DNA sequences. If the minimum set can be used to amplify the entire target DNA sequences, the experimental costs and time will be reduced. But the primer selection problem was proved to be an NP-complete problem. In this thesis, we develop an efficient heuristic algorithm for selecting a set of primer candidates, each may be able to amplify more than one target sequence. Those primers are called universal primers. The universal primer finding can be viewed as the local motif finding in our method. We modify the score function of the original Gibbs sampler method to find local motifs. The new score function is added a new parameter, weight parameter. The weight parameter can guide the Gibbs sampler method to find local motifs with the local view. Then, the complementary sequences of those local motifs are input into the binary integer programming. Thus we can reduce the size of the solution space. We also test our method on some artificial domains and two gene families. All the results show that we get some improvements on the problem.

Abstract Legionella species and Chlamydia pneumoniae are a common cause of community-acquired pneumonia and occasional cause of hospital-acquired pneumonia. Significant mortality rates among the elderly and patients with severe underlying disease may occur as a result of infection with those pathogen. Diagnostic delay may also result in increased mortality. Therefore, nucleic acid amplification assays have been shown to be useful for the detection of Legionella.spp and Chlamydia pneumoniae. The genes that encode 16S ribosomal subunits and the macrophage infectivity potentiator (MIP) gene have been shown to contain signature sequences that are useful for the identification of L. pneumophila and a variety of other Legionella species. The pst-1 fragment is useful for identification of Chlamydia pneumoniae. Here we try to test clinical specimens by Real-time PCR assays to detect L.pneumophila and other Legionella species in the same tube, and detect Chlamydia pneumoniae by SYBR Green 1 reagent. By this method, these amplicons of 16S ribosomal subunits gene and MIP gene can be discriminated by different melting curve dependent on different Tm values. In this study, we detected more 5 and 6 patients in Legionella species and Chlamydia pneumoniae than conventional diagnostic tools. Hence, the Real-time PCR also demonstrated that it¡¦s a rapid and high sensitivity method in diagnosis of legionnaires¡¦ disease. In this study, it also demonstrated that Real-time PCR is effective in prediction of atypical pathogen infection.

The aim of this thesis is to evaluate the expression of both TSG101 tumor susceptibility gene and ret oncogene in human thyroid carcinoma specimens. Functional inactivation of TSG101 in mouse fibroblast leads to cellular transformation and the ability to form metastatic tumors in nude mice. No genomic deletion of TSG101 gene has been reported in human cancer, casting a doubt on the role of TSG101 as a classical tumor suppressor. Subsequent studies reveal that TSG101 is a frequent target of spilicing defects, which is correlated with cellular stress and p53 status, and might reflect the cellular environment during the cancer development. Furthermore, recent reports demonstrate TSG101 as a part of the MDM2/p53 regulatory circuitry, a well recognized circuitry that upon deregulation results in tumorigenesis. In this study we have analyzed TSG101 gene expression in 85 specimens of thyroid carcinomas. The results indicated that 100% of papillary carcinomas (48/48), 85% of follicular carcinomas (18/21), 91% of medullary carcinomas (10/11) and 60% of undifferentiated carcinomas (3/5) showed strong to moderate cytoplasmic staining, whereas the staining was completely negative, or cytoplasmic dot-staining in the adjacent non-neoplastic follicular cells. Occasionally, the staining could be found in the nucleus. Subsequently, sequence analysis of 17 papillary carcinoma specimens revealed no mutation in steadiness box region, indicating that it might not be the cause of TSG101 protein overexpression. In summary, our results indicate strong correlation of TSG101 overexpression and thyroid carcinomas. Further experiments are urged to clarify the relationship of TSG101 overexpression and thyroid tumorigenesis. Rearrangement of ret proto-oncogene is unique to papillary thyroid carcinoma (PTC). These rearrangements consist of the fusion of ret tyrosine kinase domain to a variety of heterologous genes, thus generating chimeric transforming oncogenes termed, ret/PTC. The frequency of ret/PTC activation in non-radiation exposured adult populations has been reported to vary from 0-55% depending on the geographic distribution. To detect ret rearrangement and to identify candidate of novel ret/PTC in 62 specimens of PTC collected from southern Taiwan, a RT-multiplex PCR method was used to reveal the possible specimens that harbor ret rearrangements. Type specific-PCR amplification and subsequent sequence analysis of PCR product were performed to identify the known types of ret/PTC. We have identified two cases of ret/PTC1, two cases of ret/PTC3 and one case of ELKS-RET. Excitingly, four cases of unknown ret/PTC type were identified. Hence, 5¡¦-RACE strategy will be employed to identify novel ret/PTC in these four specimens.

Abstract This study is to elucidate the possibility of DNA fragment identification method in the forensic detection of marine microalgae (Chlorella sp. and Nannochloropsis oculata) from human (Homo sapiens) body by 3% agarose gel electrophoresis of amplification fragments via PCR-amplified ribosomal gene small subunit (SSU) rDNA molecules as primers, which are specific nucleotide segments on conserved regions (18S rDNA region (NS1-NS2 primers))on SSU rDNA and on variable regions (internal transcribed spacer (ITS) regions (primer 2/primer 5 for ITS1 region and primer 3/primer 4 for ITS2 region)). NS1-NS2-amplified PCR fragments are 550 bp for C. sp., 550 bp for N. oculata and 600 bp for human. ITS1-amplified PCR fragments are 1, 2 and 4 bands for C. sp. (350 bp), N. oculata (400 and 450 bp) and human (300, 450, 500 and 580 bp), respectively, while ITS2-amplified PCR fragments are 1, 2 and 1 bands for C. sp. (430 bp), N. oculata (430 and 600 bp) and human (400 bp), respectively. By using human-specific primers (AmpFlSTR® Profilerä PCR Amplification Kit), only human can be identified in the sample containing C. sp., N. oculata and human DNA, whereas C. sp. and N. oculata can not be detected, indicating the prevention of algal interference in human-specific primer-PCR procedures via AmpFlSTR® Profilerä PCR Amplification Kit. Detection limits of C. sp. and N. oculata DNA were 50 and 10 pg, respectively. The results of present investigation show that algae can be distinguished from human by NS1-NS2-amplified PCR fragments but not between C. sp. and N. oculata, while C. sp., N. oculata and human can be distinguished by ITS1- or ITS2-amplified PCR fragments. Evidently, the specificity of DNA segments in marine microalgal and human DNA provides the base for investigation of cause of death in drowning case in the marine environment.