Control of M-G1 phase-specific expression in fission yeast

The mitotic cell cycle underlies propagation of eukaryotic cells, continually duplicating and dividing. The past few years have seen major advances in understanding of the regulatory mechanisms that impose on the cell cycle to tightly co-ordinate progression through its individual phases, safeguarding the timing and integrity of its hallmark events, DNA synthesis and mitosis. Transcription is prominent among these processes, manifesting its importance for cell cycle controls by the large number of eukaryotic genes that are expressed at specific cell cycle times. Certain genes are cell cycle regulated in a number of organisms, suggesting that their phase-specific transcription is important for all eukaryotic cells. The budding and fission yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been used extensively as model organisms for the study of the eukaryotic cell cycle and cell cycle-regulated transcription, because the cell cycle machinery is conserved among eukaryotes and they are experimentally tractable. Recent microarray analyses have shown that cell cycle-specific expression is a frequent theme in the two yeasts, identifying consecutive, inter-dependent, waves of transcriptional activity, coinciding with the four main cell cycle transitions; G1-S, S, G2-M and M-G1 phases. Each phase-specific transcriptional wave corresponds to at least one group of co-regulated genes, sharing common cis- and trans- acting elements. The work presented in this thesis delves into the regulatory network that drives phase-specific gene expression during late mitosis-early G1 phase in fission yeast. During this late cell cycle stage, fission yeast and, indeed, every eukaryotic cell, undergo major changes; each completes mitosis and cytokinesis, partitioning its duplicated genetic and cytoplasmic material into two progeny cells, which then themselves prepare for a new round of mitotic cell division. Consistent with their periodic pattern of expression, most of the genes transcribed during the M-G1 interval in S. pombe encode proteins that execute important functions during late mitosis and cytokinesis. A DNA sequence promoter motif, the PCB (Pombe cell cycle box), has been identified in fission yeast that confers M-G1 specific transcription, and is bound by the PBF (PCB binding factor) transcription factor complex. PCB promoter motifs are present in several M-G1 transcribed genes, including cdc15+, spo12+, sid2+, fin1+, slp1+, ace2+, mid1+/dmf1+ and plo1+, the latter encoding a Polo-like kinase that also regulates M-G1 gene expression and influences the PCB-dependent binding properties of PBF. Three transcription factors, Sep1p and Fkh2p, both forkhead-like transcription factors, and Mbx1p, a MADS-box protein, have been implicated in M-G1 specific gene expression and are thought to be components of PBF. Consistent with Fkh2p and Sep1p regulating M-G1 specific transcription, forkhead-related sequences are present in the genes’ promoters. Notably, fkh2+ contains both PCB and forkhead promoter sequences and is transcribed during the M-G1 interval, implying that Fkh2p and Plo1p regulate gene transcription during late mitosis and ensuing passage through cytokinesis via feedback loops. This study provides further evidence about transcriptional regulation late in the fission yeast cell cycle, revealing that the PCB sequence is crucial for M-G1 specific transcription, with forkhead-associated DNA motifs playing a parallel but smaller regulatory role. Consistent with this hypothesis, work here and elsewhere shows that both Fkh2p and Sep1p control phase-specific expression of their co-regulated genes through the PCB and forkhead sequences. Notably, data in this thesis reveal that these two forkhead transcription factors associate with each other in vitro and in vivo and bind in vivo to the PCB promoter regions of M-G1 transcribed genes, including cdc15+ and plo1+, in a cell cycle specific manner, consistent with Fkh2p repressing and Sep1p activating transcription. Furthermore, Fkh2p contacts its own promoter, suggesting that it regulates its own expression via a negative feedback mechanism. The Plo1p kinase is shown here to bind in vivo to Mbx1p throughout the cell cycle and in a manner that requires both its kinase and polo-box domains. In agreement with this observation, Plo1p can phosphorylate in vitro Mbx1p, itself known to become phosphorylated during late mitosis. This is the first time that a Polo-like kinase has been shown to bind and phosphorylate a MADS-box protein in any organism. Moreover, in concert with Plo1p binding and phosphorylating Mbx1p, ChIP assays here reveal that this kinase interacts in vivo with the PCB promoter DNA of M-G1 expressed genes, including cdc15+ and fkh2+, in a cell cycle-dependent manner with a timing that coincides with low levels of expression, but follows promoter binding by Fkh2p. Given that Plo1p has previously been shown to positively influence M-G1 dependent transcription, its cell cycle pattern of promoter contact suggests that this Polo-like kinase functions at the genes’ promoters, most-likely via binding and phosphorylation of Mbx1p, to re-stimulate transcription, following repression by Fkh2p. In parallel, these findings suggest that Plo1p regulates its own expression via a positive feedback loop. Overall, the work presented in this thesis unravels crucial regulatory aspects of the transcriptional network that drives M-G1 specific transcription in S. pombe: it suggests an important role for the PCB promoter motif in transcriptional regulation; it proposes that Fkh2p acts as a repressor while Sep1p as an activator of late mitotic transcription; it reveals and proposes novel functions for Plo1p, a conserved Polo-like kinase family member, involving its association with Mbx1p, a MADS box protein, and its cell cycle specific recruitment to PCB promoters of M-G1 transcribed genes. As transcriptional systems, encompassing homologues of most of the components of this S. pombe M-G1 specific transcriptional network operate both in S. cerevisiae and humans, this demonstrates their importance for mitotic cell cycle progression. Thus this work potentially offers new insights into M-G1 specific gene expression in all eukaryotes

Sartorius muscle metastases from urothelial cell carcinoma

Urothelial carcinoma of the bladder represents the majority of malignant bladder tumors. Metastases to skeletal muscle from carcinoma of the bladder are extremely rare and are tend to be found in people with advanced-stage carcinoma. In this article we present a man with an unusual metastasis to the left sartorius muscle from an urothelial cell carcinoma, who was submitted in surgery and then in concomitant chemotherapy and external radiation therapy as palliative therap

Left ventricular hypertrophy induced by weight excess in children and adolescents

Background: Obesity is associated with left ventricular hypertrophy, an independent risk factor of  cardiovascular morbidity. The aim of the present study was to evaluate obesity-induced left ventricular hypertrophy in overweight/obese children and adolescents and demonstrate possible early echocardiographic abnormalities in overweight children compared to obese children.Methods: The study included 55 children and adolescents (24 overweight and 31 with  obesity) and 55 normal weight children. Standard M-mode echocardiography was performed in all participants. Parameters associated with left ventricular structure and function were recorded and statistically analysed.Results: Left ventricular hypertrophy (LVmass/height3>95th percentile) was diagnosed in 33% of overweight children and 66.5% of children with obesity (p=0.01). Mean left ventricular mass/height3 and left ventricular mass/height2.7 were significantly higher in children with obesity compared to overweight children, as well as compared to the normal weight children. On the contrary, mean values of the index left ventricular mass/body surface area did not differ significantly among study groups. Furthermore, mean left ventricular end-diastolic diameter, interventricular septal thickness and left atrial diameter, presented significant differences among the 3 studied groups, even between overweight andnormal weight children. Both systolic and diastolic function of left ventricular function were normal in our study groups.  Conclusions: Overweight children demonstrate significant early changes in left ventricular wall dimensions compared to normal weight children, while children with obesity additionally present a significant increase in left ventricular  mass. Future studies are needed to explore the effect of dietary programs and other interventions on cardiac function parameters in these children

Insulin receptor (IR) expression in human trophoblasts of recurrent pregnancy loss (RPL)

Purpose: Insulin and glucose pathways play a key role to fetal viability and growth. The focus of the study is to investigate the potential differences of immunohistochemical expression of IR in trophoblastic and decidual cells between women who had recurrent pregnancy loss and women that underwent an abortion. Materials and methods: Trophoblastic and decidual tissues from fifty (50) women with elective abortion used as control group and from fifty (50) women with recurrent miscarriages were collected during gestational weeks 6 to 12. IR antibodies were used as immunohistochemical staining markers. Nuclear and cytoplasmic expression was evaluated. Results: No IR immunohistochemical expression was detected in both trophoblastic cells of the implantation site and deciduas basalis of the two study groups. Conclusion: The effort made to enhance our knowledge on the physiology and histology of IR expression in connection with pregnancy was halted because the results were inconclusive. While studying, though, the correlation of recurrent miscarriage with IR expression, it became evident that a lot of hormones and pathways form the weave of gestational pathology and its delicate harmony. Every piece of knowledge may clarify this still obscure field

The importance of markers HLA6 and CD68 in placenta tissues of recurrent pregnancy loss

Introduction: Recurrent pregnancy loss of unknown etiology is correlated with immunological factors during pregnancy. Changes in leukocyte subpopulations and HLA (Human Leukocyte Antigen) expression take place in pregnant uterus on both decidua basalis and decidua parietalis in order to carry the semiallogenic embryo. These changes affect the pregnancy course.Objective: Our research is focused to enlighten the immunological changes that take place in the uterus of women with recurrent abortions of unknown etiology during first trimester of pregnancy.Materials and methods: The miscarriage group was obtained from 25 women who miscarried between the ages of 35 to 42 years and controls consisted of 25 healthy women between the ages of 27 to 39 years, who had electively terminated their pregnancies during first trimester of pregnancy. The abortion was processed and specimens taken were studied, using immunohistochemical methods. Specimens were taken from decidua basalis and decidua parietalis. Monoclonal antibodies were used against HLAG (Human Leukocyte Antigen G) and CD68 (Cluster of Differentiation 68). The results were statistically analysed with Mann-Whitney test.Results: HLA-G expression in decidua basalis from miscarriage group was found decreased.CD68 + cell expression was found increased in both deciduas from the miscarriage group.Conclusion: The immunological profile of women with recurrent miscarriage is quite different comparing with controls. A possible role of CD68+cells in RPL was observed .Changes in HLA-G expression was observed

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients

Background: Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). Results: Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific diseaseassociated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. Conclusions: To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance

Neuropsychological sequalae of coronary artery bypass grafting.

Objectives: To assess neurocognitive function after Coronary Artery Bypass Grafting (CABG); to compare and contrast pre- and post -operative test results and to characterise the degree of change in neurocognitive profiles.To identify neuropsychological patterns associated with different impairment models such as hypoxia and stroke. Methods: A comprehensive neuropsychological battery of tests was administered to a small number of individuals undergoing CABG at pre and post operative stage. In order to minimise practice effects, alternate tests were employed for memory, verbal and visuo-spatial functions. Mood measures were included. Results: There were six variables that indicated significant changes. These changes were in Processing Motor Speed, Verbal Executive function, Verbal, Visual learning and Memory, Verbal functions and Visuospatial functions. There was an overall improvement on mood after CABG. Conclusions: There were different neuropsychological sequelae -considering hypoxia and stroke impairment models- in the participants undergoing CABG via Cardiopulmonary Bypass (CPB). Neuropsychological deficits and improvements were identified

Digital Technologies in Museums : An ethnographic study

This thesis follows a research at the Mineralogy and Petrology Museum of the University of Athens (Greece). The purpose of the research is to examine how digital technologies can support museums in attracting new visitors during the economic crisis, and provide design suggestions. The research was conducted under the interpretive paradigm using the ethnographic methodology and the participatory design approach. The staff, volunteers and artists related to the museum participated in interviews, thinking aloud sessions and a workshop. In addition, several observations were conducted during guided tours of visitors. The data were analyzed through thematic analysis. The findings provided themes of identified issues and opportunities concerning the implementation of digital technologies in the museum, such as the underlying policies of the University of Athens, the role of the museum in society, material in the collections that attracts visitors, the importance of the staff in guided tours and the technologies currently being used. Suggestions were put forward during the workshop and were elaborated later on. The thesis aspires to contribute to studies that concern the sustainability of cultural establishments that are being afflicted by the economic crisis currently veils Europe.

Digital Technologies in Museums : An ethnographic study

This thesis follows a research at the Mineralogy and Petrology Museum of the University of Athens (Greece). The purpose of the research is to examine how digital technologies can support museums in attracting new visitors during the economic crisis, and provide design suggestions. The research was conducted under the interpretive paradigm using the ethnographic methodology and the participatory design approach. The staff, volunteers and artists related to the museum participated in interviews, thinking aloud sessions and a workshop. In addition, several observations were conducted during guided tours of visitors. The data were analyzed through thematic analysis. The findings provided themes of identified issues and opportunities concerning the implementation of digital technologies in the museum, such as the underlying policies of the University of Athens, the role of the museum in society, material in the collections that attracts visitors, the importance of the staff in guided tours and the technologies currently being used. Suggestions were put forward during the workshop and were elaborated later on. The thesis aspires to contribute to studies that concern the sustainability of cultural establishments that are being afflicted by the economic crisis currently veils Europe.

The Role of Epikarst in the Morphogenesis of the Karstic Forms, Especially Hollow Forms in Greece

Vloga epikrasa pri nastanku podtalnih škrapelj in kraških depresij, je različna. Prevotljene škraplje in podtalne škraplje tipa »kluftkarren« so prekrite s kraško prstjo in kažejo na delovanje vode. Nastale so v epikraški coni ob razpokah s pomočjo difuzne korozije v kapilarnem vodonosniku v zgornjem delu vadozne cone. Zaobljeni vrhnji deli in luknje v škrapljah so nastale zaradi enakomerne in stalne korozije s pomočjo talne vlage v zgornjem delu epikraške cone. Kraške depresije pa so razvite v karbonantih plasteh in tudi na stiku zakraselih in nezakraselih, a eroziji podvrženih kamnin. V tem primeru nastane veliko drobirja, iz katerega nastane prst, ki zapolni te reliefne oblike. Uvale in polja so navadno razvita na mestu nekdanjih suhih dolin ali tektonskih udorin s pomočjo razširjanja razpok v subkutani coni in zaradi njenega spuščanja. Sočasno ta cona postaja postopoma neprepustna in s tem se krepi robna korozija. Te oblike navadno nastajajo nad piezometrično gladino. Kraška polja – presihajoča jezera pa so nastala, kjer so depresije občasno dosegle gladino talne vode v epikrasu. The role of epikarst in the morphogenesis of the subcutaneous karren and the karstic hollow forms is different. Cavernous karren and subcutaneous kluftkarren, are covered with terra rossa and show the activity of water. They are developed in the epikarstic zone due to the diffused corrosion on the joints through a capillary aquifer, situated in the high vadose zone. The rounded tops and the cavities of those subcutaneous karren are formed due to the continuous and uniform corrosion, acting by soil moisture in the high part of the epikarstic zone. Karstic hollowforms are developed in carbonate layers only, and also on the contact of karstifi ed and non-karstifi ed but easily erodable rocks. In this case, a lot of material is produced for the formation of soils that fi ll these landforms. Uvalas and poljes usually develop in sites of old valleys or tectonic depressions through the process of widening of the joints in the subcutaneous zone and the lowering of the latter zone. Simultaneously there occurs the gradual impermeability of the zone and the reinforcement of the lateral corrosion. These forms are mostly formed above the piezometric level. The poljes - periodical lakes are created due to the development when they temporary reach epikarstic water table