Tato diplomová práce se zaměřuje na popis specifik práce rozhlasového sportovního redaktora/komentátora. teoretické části popisuje historii vzniku samotného Československého rozhlasu a rozhlasové sportovní reportáže, která je spojená se jménem vůbec prvního československého rozhlasového sportovního reportéra Josefa Laufera. Druhá část je zaměřená na terminologické vymezení pojmů rozhlasový sportovní redaktor, reportér a komentátor. Dále na základě rozhovorů tohoto oboru nabízí náhled do zákulisí této práce včetně jejích charakteristik a specifik od samotného přijetí do sportovní Redakce Českého rozhlasu, předpokladů pro její vykonávání až po práci v terénu a redakci. Závěrečná část analyzuje rozhlasovou reportáž komentátora Aleše Procházky z finálového zápasu hokejového turnaje na zimních olympijských hrách v 1998 mezi Českem a Ruskem.

Proteases are crucial to many biological processes and have become an important field of biomedical and biotechnological research. Engineering of proteases towards therapeutic applications has been limited due to the lack of high-throughput methods for characterization and selection. We have developed a novel high-throughput method for quantitative assessment of proteolytic activity in the cytoplasm of Escherichia coli bacterial cells. The method is based on coexpression of a protease of interest and a reporter complex consisting of an aggregation-prone protein fused to a fluorescent reporter. Cleavage of a substrate sequence situated between the two reporter complex proteins results in increased whole-cell fluorescence proportional to proteolytic activity, which can be monitored using flow cytometry. We have demonstrated that the method can distinguish efficiencies with which Tobacco Etch Virus (TEV) protease processes different substrates. We believe that this is the first method in the field of protease engineering that enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for substrate profiling, as well as screening and selection of libraries of engineered proteases.

Reporter genes are often used to observe expression of promoters, which may change from its natural behaviour as a result of stress or disease states. Reporter genes are useful because they are easily detectable by a variety of imaging methods, including fluorescence microscopy techniques, magnetic resonance imaging, and positron emission tomography. Previously, methyl 5-MeO-N-aminoanthranilate (MMNA) had been synthesized as an aldehyde-conditional fluorophore and was tested in physiological conditions to identify the Aldehydic Load of cells. Thus, it was hypothesized that a reporter protein displaying an aldehyde on the cell surface can be identified by MMNA. This reporter protein would contain a substrate recognition site for formylglycine generating enzyme (FGE) that converts a specific cysteine residue into a formylglycine residue. This will result in production of an aldehyde at the N-terminal of the transmembrane domain of platelet derived growth factor receptor . In this way, the protein product, Aldehyde-presenting FGE-dependent Readout (Alfred), would display an aldehyde on the extracellular surface of the cell. Alfred was expressed in A549 human lung cancer cells using the Tet-On® Inducible System, which allows expression of a gene of interest by use of doxycyclin (dox) as a chemical trigger. Microscopy of Alfred-transfected cells, induced by dox and probed with MMNA, showed no difference in fluorescence between non-transfected and Alfred-transfected cells. The overexpression of FGE to increase thiol-to-aldehyde conversion, and the imaging of cells at longer timepoints (48 and 72 hours) to allow localization of the protein to the cell surface, were attempted. In addition, Alfred was constitutively expressed in another transfection experiment in efforts to increase gene expression. However, these efforts to evaluate Alfred did not improve the microscopy results. Western blotting confirmed FGE overexpression in transgenic cells. Blotting against the Myc-tag in Alfred showed no detected proteins in Alfred-transfected cells. In conjunction with the microscopy images, these results suggest that Alfred is not expressed and cannot be detected as a reporter gene. Comparison to previous works allows the identification of potential approaches to improve Alfred functionality, including the absence of the hemagglutinin epitope, the choice of aldehyde probe used, the choice of cell line used, and the method of analyzing microscopy. Future directives are postulated to identify sources that hinder Alfred expression, and to improve visualization of Alfred over homeostatic aldehydes.

The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development. Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our  findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues. / Master of Science

Final publication is available at http://dx.doi.org/10.1016/j.stemcr.2014.07.005. Shimpei Gotoh, Isao Ito, Tadao Nagasaki, Yuki Yamamoto, Satoshi Konishi, Yohei Korogi, Hisako Matsumoto, Shigeo Muro, Toyohiro Hirai, Michinori Funato, Shin-Ichi Mae, Taro Toyoda, Aiko Sato-Otsubo, Seishi Ogawa, Kenji Osafune, Michiaki Mishima, Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells, Stem Cell Reports, Volume 3, Issue 3, 9 September 2014, Pages 394-403, ISSN 2213-6711. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18681号 / 医博第3953号 / 新制||医||1007(附属図書館) / 31614 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 江藤 浩之, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis advisor: Vassilios Bezzerides / The aim of this study was to utilize a massively parallel reporter assay (MPRA) to identify organ-specific microRNA (miRNA) target sequences to refine the timing and expression of transgene expression for gene therapy. We previously had developed a cardiac gene therapy for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) using a systemically delivered adeno-associated virus (AAV9) vector. We hypothesized that incorporation of organ specific miRNA target sites into our vector construct could improve our therapy’s tissue specificity due to the ability of miRNAs to silence transgene expression. Initially, we attempted to incorporate mir-124 target sequences into our vector to detarget the brain. Although these initial attempts were unsuccessful, the study allowed us to develop a protocol to test the effectiveness of miRNA target sequences. Thereafter, we developed a method to screen thousands of putative miRNA target sequences simultaneously. In this study, target sequences of miRNAs specific to the heart, brain and liver were incorporated into a plasmid library. This plasmid library was subsequently made into AAV and injected into mice from a CPVT transgenic line. Total DNA and RNA was later extracted from the target organs, converted into genomic DNA (gDNA) and complementary DNA (cDNA) libraries respectively, and sent for amplicon sequencing. We analyzed the results using Comparative Microbiome Analysis 2.0 software (CoMA) and a custom python script to count the occurrence of each specified barcode per sample. In doing so, we showed that the miRNA suppression mechanism is not only effective but also organ specific. Furthermore, we developed a second script to create a combinatorial library from a set list of miRNA target sequences enabling us to efficiently test thousands of target sequence combinations at once. In doing so, we will be able to identify effective miRNA target sequence combinations to further improve gene therapy specificity. / Thesis (BS) — Boston College, 2021. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.

Osteoarthritis is a joint disease characterized by the breakdown of articular cartilage. The field of tissue engineering is interested in developing methods to produce biological alternatives to current orthopedic procedures. Lubricin is a molecule which is important in the proper lubrication of articular cartilage. It is a challenge in the field of tissue engineering to produce cartilage with sufficient lubricin expression. Developing a reporter cell for lubricin allowed for a more efficient investigation of the conditions wh­­­ich may influence its expression. By comparing "optimized" and traditional media solutions, it was determined that the use of a previously reported type II collagen optimized media would negatively affect the expression of lubricin. This information indicates the need to further evaluate the conditions which are conducive to producing cartilage with both sufficient types of type II collagen and lubricin.

京都大学 / 新制・論文博士 / 博士(人間健康科学) / 乙第13562号 / 論人健博第12号 / 新制||人健||8(附属図書館) / (主査)教授 高桑 徹也, 教授 藤井 康友, 教授 長尾 美紀 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Uppsatsen studerar två nutida reportage i reportagebokens format: Klubben av Matilda Gustavsson, och Smärtpunkten av Elisabeth Åsbrink. Syftet är att undersöka hur reportern själv och det journalistiska arbetet framställs i reportagen och vad det får för konsekvenser för reportagens journalistiska syften. I uppsatsen tillämpas narratologisk teori utifrån begrepp som modus, tempus, röst, konsonans och dissonans. Även journalistisk teori om objektivitet och transparens, samt teorier om trovärdighet, autenticitet och life writing används. Båda reportagen har en komplex narratologisk konstruktion som innehåller en subjektivt jag-berättande reporter och en ramberättelse som detaljerat beskriver reporterns eget journalistiska arbete. Analysen visar hur konstruktionen med ramberättelsen om det journalistiska arbetet och det subjektiva jag-berättandet skapar trovärdighet, närvaro och engagemang för reportagets ärende. I synnerhet i Klubben används det subjektiva jag-berättandet som en strategi som stämmer väl överens med teorierna om life writing. Detta framhäver reportagets vittnande karaktär, där både reportern och de karaktärer som hon berättar om framstår som röster och vittnesbörd i en större berättelse om förövare och offer, men också i berättelsen om journalistikens viktiga roll att granska samhället och avslöja missförhållanden. Det subjektiva berättandet, med sina drag av life writing, bidrar alltså till engagemang, autenticitet, trovärdighet och transparens i beskrivningarna av både de händelser som reportagen beskriver och av det journalistiska arbetet i sig.