New Method for Delexicalization and its Application to Prosodic Tagging for Text-to-Speech Synthesis

This paper describes a new flexible delexicalization method based on glottal excited parametric speech synthesis scheme. The system utilizes inverse filtered glottal flow and all-pole modelling of the vocal tract. The method provides a possibil- ity to retain and manipulate all relevant prosodic features of any kind of speech. Most importantly, the features include voice quality, which has not been properly modeled in earlier delex- icalization methods. The functionality of the new method was tested in a prosodic tagging experiment aimed at providing word prominence data for a text-to-speech synthesis system. The ex- periment confirmed the usefulness of the method and further corroborated earlier evidence that linguistic factors influence the perception of prosodic prominence.

Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.