Evaluation von Effektivität und Sicherheit der niederfrequenten, transkraniellen Ultraschalltherapie beim Schlaganfall

In den westlichen Ländern nimmt die Zahl der Schlaganfall-Patienten stetig zu und zählt mittlerweilernzu einer der häufigsten Todesursachen. Derzeit ist die Rekanalisationstherapie mit demrnFibrinolytikum rt-PA die einzig zugelassene Therapie. Die Rekanalisationsrate ist oftmals inkomplettrnund aufgrund von möglichen Blutungskomplikationen die Therapie nicht bei allen Patientenrnmöglich. Daher ist es wichtig, Alternativtherapieansätze (z.B. Ultraschallthrombolyse) zurnentwickeln. Blutgerinnsel können mit Hilfe von Ultraschall in Schwingung gebracht und sornlysiert oder die Wirkung von rt-PA verstärkt werden. Die vorliegende Arbeit hatte die Evaluationrnvon Bioeffekten von 60 kHz Ultraschall an gesundem und ischämischem Hirngewebe zum Ziel.rnNeben tierexperimentellen Methoden kamen auch molekular-biologische Techniken zur Anwendung.rnDie erste Studie beschäftigte sich mit der Wirkung von 60 kHz (Intensität: 0,2 W/cm2 undrnDuty Cycle 50%) auf ischämisches Hirngewebe (permanent ischämisch und nach Reperfusion).rnLediglich nach Reperfusion und Ultraschallbehandlung war das Läsionsvolumen signifikantrnerhöht, so dass von einer besonderen Vulnerabilität des Hirngewebes nach Reperfusionrnauszugehen ist (Penumbraschädigung). In der neurologischen Beurteilung der Tiere zeigte sichrnbei allen Tieren mit permanenter Okklusion und etwa einem Drittel der Tiere nach Reperfusionrnund Ultraschallbehandlung eine Hörminderung. In der anschließenden Studie wurde diernUltraschallintensität erniedrigt und der Duty Cycle variiert. In einer publizierten in vitro Studiernkonnte die zunehmende Lyserate mit steigendem Duty Cycle nachgewiesen werden. DiernAuswertung ergab eine Abhängigkeit des Läsionsvolumens von der Länge des Duty Cycles. Derrndritte Teil der Arbeit befasst sich mit der Wirkung von Ultraschall auf die Genexpression. Hierzurnwurden gesunde Ratten mit Ultraschall verschiedener Frequenzen (60 kHz, 488 kHz und 3 MHz)rntranskraniell behandelt und 4 h bzw. 24 h nach der Behandlung getötet. Proben von ischämischenrnTieren dienten als positive Kontrollen. Aufgrund von Literaturrecherchen wurden mehrerernKandidatengene ermittelt. Die Messung der Ischämieproben ergab eine weitgehende Übereinstimmungrnmit der Literatur. Die Messungen an den mit 60 kHz behandelten Proben ergabenrnkaum Anzeichen für eine differenzielle Genregulation. Die Frequenz von 488 kHz zeigte diernmeisten Regulationen, gefolgt von der Behandlung mit 3 MHz. Dieses Ergebnis lässt vermuten,rndass es sich bei den detektierten Veränderungen um protektive Mechanismen handelt, da diesernFrequenzen bislang im Tierversuch als nebenwirkungsarm beschrieben wurden. Die Auswertungrnvon 60 kHz-Proben mit Affymetrix Arrays ergab lediglich einige wenige differentiell regulierternGene. Die Array-Experimente konnten nicht durch qPCR-Messungen bestätigt werden.

Expression of estrus modifies the gene expression profile in reproductive tissues on Day 19 of gestation in beef cows

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Cluster-based network model for time-course gene expression data

We propose a model-based approach to unify clustering and network modeling using time-course gene expression data. Specifically, our approach uses a mixture model to cluster genes. Genes within the same cluster share a similar expression profile. The network is built over cluster-specific expression profiles using state-space models. We discuss the application of our model to simulated data as well as to time-course gene expression data arising from animal models on prostate cancer progression. The latter application shows that with a combined statistical/bioinformatics analyses, we are able to extract gene-to-gene relationships supported by the literature as well as new plausible relationships.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Nuclear localization of NPR1 is required for activation of PR gene expression

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1–green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

Insulin-like growth factor-i : vitronectin complex-induced changes in gene expression effect breast cell survival and migration

Recent studies have demonstrated that IGF-I associates with VN through IGF-binding proteins (IGFBP) which in turn modulate IGF-stimulated biological functions such as cell proliferation, attachment and migration. Since IGFs play important roles in transformation and progression of breast tumours, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function and to dissect mechanisms underlying these responses. In this study we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival, which is mediated by a transient activation of ERK/MAPK and sustained activation of PI3-K/AKT pathways. Furthermore, use of pharmacological inhibitors of the MAPK and PI3-K pathways confirms that both pathways are involved in IGF-I:IGFBP:VN complex-mediated increased cell survival. Microarray analysis of cells stimulated to migrate in response to IGF-I:IGFBP:VN complexes identified differential expression of genes with previously reported roles in migration, invasion and survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1). These changes were not detected when the IGF-I analogue (\[L24]\[A31]-IGF-I), which fails to bind to the IGF-I receptor, was substituted; confirming the IGF-I-dependent differential expression of genes associated with enhanced cell migration. Taken together, these studies have established that IGF-I:IGFBP:VN complexes enhance breast cell migration and survival, processes central to facilitating metastasis. This study highlights the interdependence of ECM and growth factor interactions in biological functions critical for metastasis and identifies potential novel therapeutic targets directed at preventing breast cancer progression.