Sequential incoherence in a multi-party synchronous computer mediated communication for an introductory Health Informatics course.

Online courses will play a key role in the high-volume Informatics education required to train the personnel that will be necessary to fulfill the health IT needs of the country. Online courses can cause feelings of isolation in students. A common way to address these feelings is to hold synchronous online "chats" for students. Conventional chats, however, can be confusing and impose a high extrinsic cognitive load on their participants that hinders the learning process. In this paper we present a qualitative analysis that shows the causes of this high cognitive load and our solution through the use of a moderated chat system.

STUDIES ON THE BIOLOGICAL ACTIVITY OF MACROMOLECULES MICROINJECTED INTO MAMMALIAN SOMATIC CELLS

Red Blood cell mediated and glass needle mediated microinjection technology was used to introduce macromolecules into mammalian somatic cells. The biological activities of DNA synthesis inducing factor(s) (Chapter 1), mitotic factor(s) (Chapter 2), and DNA coding for ovalbumin and thymidine kinase (Chapter 3) were studied following injection into mammalian somatic cells.^ Chapter 1. A cell undergoing DNA replication (S phase) contains a factor(s) that induces DNA synthesis prematurely in a G(,1) nucleus when an S phase cell is fused to a G(,1) cell. An assay for the active factor(s) was developed in which a mixture of s phase extract loaded red blood cells (RBC) and synchronous G(,1) HeLa cells was centrifuged onto Concanavalin A (Con A) treated coverslips and fused by PEG. This technique is called "Centrifusion". The synchronous G(,1) HeLa cells injected with S phase extract initiated DNA synthesis earlier than the control G(,1) cells mock injected with RBC loaded with buffer.^ Chapter 2. It has been demonstrated that fusion between a mitotic and an interphase cell usually leads to breakdown of the interphase nucleus, followed by condensation of the interphase chromatin into discrete chromosomes, a process termed premature chromosome condensation. I wanted to develop an assay for the mitotic factor(s) that induces premature chromosome condensation. Experiments were performed utilizing glass needle mediated microinjection of HeLa cell mitotic extract into interphase somatic mammalian cells in an attempt to induce premature chromosome condensation. However, I was not able to induce premature chromosome condensation in the interphase cells, probably because of an inability to introduce sufficient mitotic factor(s) into the cells.^ Chapter 3. A recombinant plasmid containing the chicken ovalbumin gene and three copies of the Herpes thymidine Kinase gene (pOV12-TK) was introduced into mouse LMTK('-) cell nuclei using glass needle mediated gene transfer resulting in LMTK('+) clones that were selected for in HAT medium. Restriction enzyme analysis of the high molecular weight DNA from 6 HAT medium survivor cell clones revealed the presence of one or at best only a few copies of the 12kb ovalbumin gene per mouse genome. Further analysis showed the ovalbumin DNA was not rearranged and was associated with high molecular weight mouse cell DNA. Each of the analyzed cell clones produced ovalbumin demonstrating that the biological activity of the microinjected ovalbumin was retained. ^

Expression of p53, {\it * ras\/} and PCNA in human colonic neoplasms: Possible biomarkers of malignant potential

Colorectal cancer is a leading cause of cancer mortality and early detection can significantly improve the clinical outcome. Most colorectal cancers arise from benign neoplastic lesions recognized as adenomas. Only a small percentage of all adenomas will become malignant. Thus, there is a need to identify specific markers of malignant potential. Studies at the molecular level have demonstrated an accumulation of genetic alterations, some hereditary but for the most occurring in somatic cells. The most common are the activation of ras, an oncogene involved in signal transduction, and the inactivation of p53, a tumor suppressor gene implicated in cell cycle regulation. In this study, 38 carcinomas, 95 adenomas and 20 benign polyps were analyzed by immunohistochemistry for the abnormal expression of p53 and ras proteins. An index of cellular proliferation was also measured by labeling with PCNA. A general overexpression of p53 was immunodetected in 66% of the carcinomas, while 26% of adenomas displayed scattered individual positive cells or a focal high concentration of positive cells. This later was more associated with severe dysplasia. Ras protein was detected in 37% of carcinomas and 32% of adenomas mostly throughout the tissue. p53 immunodetection was more frequent in adenomas originating in colons with synchronous carcinomas, particularly in patients with familial adenomatous polyposis and it may be a useful marker in these cases. Difference in the frequency of p53 and ras alterationbs was related to the location of the neoplasm. Immunodetection of p53 protein was correlated to the presence of a mutation in p53 gene at exon 7 and 5 in 4/6 carcinomas studied and 2 villous adenomas. Thus, we characterized in adenomas the abnormal expression of two proteins encoded by the most commonly altered genes in colorectal cancer. p53 alteration appears to be more specifically associated with transition to malignancy than ras. By using immunohistochemistry, a technique that keeps the architecture of the tissue intact, it was possible to correlate these alterations to histopathological characteristics that were associated with higher risks for transformation: villous content, dysplasia and size of adenoma. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^