Drop formation in non-newtonian liquids under pulsed conditions

Drop formation from single nozzles under pulsed flow conditions in non-Newtonian fluids following the power law model has been studied. An existing model has been modified to explain the experimental data. The flow conditions employed correspond to the mixer—settler type of operation in pulsed sieve-plate extraction columns. The modified model predicts the drop sizes satisfactorily. It has been found that consideration of non-Newtonian behaviour is important at low pulse intensities and its significance decreases with increasing intensity of pulsation. Further, the proposed model for single orifices has been tested to predict the sizes of drops formed from a sieve-plate distributor having four holes, and has been found to predict the sizes fairly well in the absence of coalescence.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

Proteolytic susceptibility and methionine modification of monodeamidated ribonuclease A

The susceptibility of a monodeamidated RNAaseA (RNAaseAa1) towards carboxypeptidaseA , alpha-chymotrypsin and pepsin has been studied. Similar to RNAaseA, the C-terminal of RNAaseAa1 is not available for carboxypeptidaseA hydrolysis. The thermal stability of RNAaseAa1 as probed through chymotryptic digestion is found to be less than that of RNAaseA. Preliminary chromatographic analysis of the digested material, however, suggests that the nature of thermal transition might be the same in the two proteins. Pepsin inactivates RNAaseAa1 more slowly than does RNAaseA. Accordingly, less peptide bonds, almost half that of RNAaseA, are cleaved by pepsin in RNAaseAa1. The accumulation of RNAase-P type intermediates is not evident during peptic digestion of RNAaseAa1. Reaction with O-benzoquinone at low pH shows that methionines of the deamidated protein seem to have higher reactivities. These observations indicate a different structure for RNAaseAa1 at elevated temperature and low pH.

The neuronal cell adhesion molecule ICAM-5

The neuronal cell adhesion molecule ICAM-5 ICAM-5 (telencephalin) belongs to the intercellular adhesion molecule (ICAM)-subgroup of the immunoglobulin superfamily (IgSF). ICAMs participate in leukocyte adhesion and adhesion-dependent functions in the central nervous system (CNS) through interacting with the leukocyte-specific b2 integrins. ICAM-5 is found in the mammalian forebrain, appears at the time of birth, and is located at the cell soma and neuronal dendrites. Recent studies also show that it is important for the regulation of immune functions in the brain and for the development and maturation of neuronal synapses. The clinical importance of ICAM-5 is still under investigation; it may have a role in the development of Alzheimer s disease (AD). In this study, the role of ICAM-5 in neuronal differentiation and its associations with a-actinin and N-methyl-D-aspartic acid (NMDA) receptors were examined. NMDA receptors (NMDARs) are known to be involved in many neuronal functions, including the passage of information from one neuron to another one, and thus it was thought important to study their role related to ICAM-5. The results suggested that ICAM-5 was able to induce dendritic outgrowth through homophilic adhesion (ICAM-5 monomer binds to another ICAM-5 monomer in the same or neighbouring cell), and the homophilic binding activity appeared to be regulated by monomer/multimer transition. Moreover, ICAM-5 binding to a-actinin was shown to be important for neuritic outgrowth. It was examined whether matrix metalloproteinases (MMPs) are the main enzymes involved in ICAM-5 ectodomain cleavage. The results showed that stimulation of NMDARs leads to MMP activation, cleavage of ICAM-5 and it is accompanied by dendritic spine maturation. These findings also indicated that ICAM-5 and NMDA receptor subunit 1 (NR1) compete for binding to a-actinin, and ICAM-5 may regulate the NR1 association with the actin cytoskeleton. Thus, it is concluded that ICAM-5 is a crucial cell adhesion molecule involved in the development of neuronal synapses, especially in the regulation of dendritic spine development, and its functions may also be involved with memory formation and learning.

A simple model for protein thermal denaturation

Whether proteins denature in all-or-none fashion or in a continuous fashion is as yet an unresolved problem. The all-or-none process implies that while the process of denaturation is going on, only two kinds of protein molecules can exist. One is completely unchanged and the other is altered. The altered protein molecules are indistinguishable. Underlying the 'continuum' models is the assumption that all the chains in a protein globule undergo similar changes so that it is enough to consider a single chain.

Studies of the Human Transcriptome

Gene expression is one of the most critical factors influencing the phenotype of a cell. As a result of several technological advances, measuring gene expression levels has become one of the most common molecular biological measurements to study the behaviour of cells. The scientific community has produced enormous and constantly increasing collection of gene expression data from various human cells both from healthy and pathological conditions. However, while each of these studies is informative and enlighting in its own context and research setup, diverging methods and terminologies make it very challenging to integrate existing gene expression data to a more comprehensive view of human transcriptome function. On the other hand, bioinformatic science advances only through data integration and synthesis. The aim of this study was to develop biological and mathematical methods to overcome these challenges and to construct an integrated database of human transcriptome as well as to demonstrate its usage. Methods developed in this study can be divided in two distinct parts. First, the biological and medical annotation of the existing gene expression measurements needed to be encoded by systematic vocabularies. There was no single existing biomedical ontology or vocabulary suitable for this purpose. Thus, new annotation terminology was developed as a part of this work. Second part was to develop mathematical methods correcting the noise and systematic differences/errors in the data caused by various array generations. Additionally, there was a need to develop suitable computational methods for sample collection and archiving, unique sample identification, database structures, data retrieval and visualization. Bioinformatic methods were developed to analyze gene expression levels and putative functional associations of human genes by using the integrated gene expression data. Also a method to interpret individual gene expression profiles across all the healthy and pathological tissues of the reference database was developed. As a result of this work 9783 human gene expression samples measured by Affymetrix microarrays were integrated to form a unique human transcriptome resource GeneSapiens. This makes it possible to analyse expression levels of 17330 genes across 175 types of healthy and pathological human tissues. Application of this resource to interpret individual gene expression measurements allowed identification of tissue of origin with 92.0% accuracy among 44 healthy tissue types. Systematic analysis of transcriptional activity levels of 459 kinase genes was performed across 44 healthy and 55 pathological tissue types and a genome wide analysis of kinase gene co-expression networks was done. This analysis revealed biologically and medically interesting data on putative kinase gene functions in health and disease. Finally, we developed a method for alignment of gene expression profiles (AGEP) to perform analysis for individual patient samples to pinpoint gene- and pathway-specific changes in the test sample in relation to the reference transcriptome database. We also showed how large-scale gene expression data resources can be used to quantitatively characterize changes in the transcriptomic program of differentiating stem cells. Taken together, these studies indicate the power of systematic bioinformatic analyses to infer biological and medical insights from existing published datasets as well as to facilitate the interpretation of new molecular profiling data from individual patients.

Transformations of acetates of citronellol, geraniol, and linalool by Aspergillus niger: regiospecific hydroxylation of citronellol by a cell-free system

Incubation of acetates of geraniol, citronellol and linalool with Aspergillus niger resulted in their hydrolysis to corresponding alcohols which were further hydroxylated to their respective 8-hydroxy derivatives. In the case of linalyl acetate, besides linalool and 8-hydroxylinalool, small amounts of geraniol and agr-terpineol were also formed. Microsomes (105 000xg sediment) prepared from induced cells of A. niger were found to convert (1-3H)citronellol to 8-hydroxy citronellol in the presence of NADPH and O2. The pH optimum for the hydroxylase was found to be 7.6.

Integration of miRNA and mRNA expression with DNA copy number of Ewing sarcoma cell lines

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

Effect of follicle-stimulating hormone and its antiserum on the activity of ornithine decarboxylase in the ovary of rat and hamster

The ability of FSH to stimulate the activity of ornithine decarboxylase (ODC) in the ovary of the immature rat and cycling hamster has been examined using specific antisera to gonadotropins. The stimulatory effect of FSH on ODC activity in the ovary of the immature rat was abolished when LH antiserum was administered along with FSH, while similar administration of FSH antiserum had no effect on LH action in stimulating ODC activity, thereby demonstrating the specificity of the LH effect. During the estrus cycle of the hamster, ODC activity in the ovary could be detected only on the evening of proestrus, the maximal activity seen at 1700 h being associated with both the Graafian follicles and the rest of the ovarian tissue. Neutralization of the proestrous FSH surge had no effect on the activity of ODC in either of these tissues, while similar administration of LH antiserum at 1300 h of proestrus completely inhibited the ODC activity in both large follicles and the rest of the ovarian tissue. Thus, the surge of LH, but not of FSH, appears to be responsible for regulating the ODC activity in the ovary of the cycling hamster.

Thermal reactivity of ammonium perchlorate crystallised from solutions having different pH

The thermal reactivity of ammonium perchlorate was found to be dependent on the pH of the solution from which it had been crystallised. A nitric acid-crystallised sample reacted faster than an ammonium hydroxide-crystallised one.

Mammalian Mat1 subunit of Transcription Factor IIH in gene-specific and general transcription

All protein-encoding genes in eukaryotes are transcribed into messenger RNA (mRNA) by RNA Polymerase II (RNAP II), whose activity therefore needs to be tightly controlled. An important and only partially understood level of regulation is the multiple phosphorylations of RNAP II large subunit C-terminal domain (CTD). Sequential phosphorylations regulate transcription initiation and elongation, and recruit factors involved in co-transcriptional processing of mRNA. Based largely on studies in yeast models and in vitro, the kinase activity responsible for the phosphorylation of the serine-5 (Ser5) residues of RNAP II CTD has been attributed to the Mat1/Cdk7/CycH trimer as part of Transcription Factor IIH. However, due to the lack of good mammalian genetic models, the roles of both RNAP II Ser5 phosphorylation as well as TFIIH kinase in transcription have provided ambiguous results and the in vivo kinase of Ser5 has remained elusive. The primary objective of this study was to elucidate the role of mammalian TFIIH, and specifically the Mat1 subunit in CTD phosphorylation and general RNAP II-mediated transcription. The approach utilized the Cre-LoxP system to conditionally delete murine Mat1 in cardiomyocytes and hepatocytes in vivo and and in cell culture models. The results identify the TFIIH kinase as the major mammalian Ser5 kinase and demonstrate its requirement for general transcription, noted by the use of nascent mRNA labeling. Also a role for Mat1 in regulating general mRNA turnover was identified, providing a possible rationale for earlier negative findings. A secondary objective was to identify potential gene- and tissue-specific roles of Mat1 and the TFIIH kinase through the use of tissue-specific Mat1 deletion. Mat1 was found to be required for the transcriptional function of PGC-1 in cardiomyocytes. Transriptional activation of lipogenic SREBP1 target genes following Mat1 deletion in hepatocytes revealed a repressive role for Mat1apparently mediated via co-repressor DMAP1 and the DNA methyltransferase Dnmt1. Finally, Mat1 and Cdk7 were also identified as a negative regulators of adipocyte differentiation through the inhibitory phosphorylation of Peroxisome proliferator-activated receptor (PPAR) γ. Together, these results demonstrate gene- and tissue-specific roles for the Mat1 subunit of TFIIH and open up new therapeutic possibilities in the treatment of diseases such as type II diabetes, hepatosteatosis and obesity.