The role of imprinting in embryonic development and tumorigenesis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2006.Includes bibliographical references (v. 2, leaves 150-197).Imprinting is a mammalian adaptation that results in the mono-allelic expression of a subset of genes depending on their parental origin. It is believed that DNA methylation marks are responsible for maintaining imprinted gene expression patterns. The 'parental conflict' hypothesis was proposed to explain the evolution of imprinting and is based on the assumption that mammals arose from an ancestor that was polyandrous (multiple fathers within one litter). According to this hypothesis, conflict between the male and female over the allocation of maternal resources to the offspring led to the evolution of imprinting. Consistent with this, many imprinted genes are involved in embryonic or placental growth by regulating mitogenic pathways or the cell cycle. Loss of imprinting (LOI) has been found at specific loci in cancers, raising the possibility that altered expression of imprinted genes may also contribute to tumorigenesis. To investigate the effect of global LOI on embryonic development and cancer formation, imprint free (IF) embryonic stem (ES) cells were generated using conditional inactivation/reactivation of the DNA methyltransferase Dnmtl. Tetraploid complementation and chimera experiments revealed that IF-embryos fail to develop beyond E11.5 and display an overgrowth phenotype.(cont.) As developmental comparisons, parthenogenetic and androgenetic (AT) embryos were derived and found to develop to E9.5 and E7.5, respectively. Removing the imprinted methyl marks from AT-ES cells rescued embryonic development to E9.5-10.5 and restored pluripotency. However, IFAT embryos were not developmentally equivalent to biparentally-derived IF-embryos, suggesting that mechanisms other than DNA methylation may be involved in maintaining parent-specific gene expression patterns. To study the effect of LOI on cell growth, murine embryonic fibroblasts (MEFs) were derived from E13.5 chimeric IF-embryos and analyzed in vitro. IF-MEFs grew faster, were resistant to the cytostatic effects of TGFI3, and formed tumors in SCID mice. In addition, IF-MEFs were immortal and when exposed to H-Ras became fully transformed. Western blot analysis of IF-MEFs revealed abnormally low levels of p19Arf and p53, two critical regulators of growth arrest and potent tumor suppressors. Somatic contribution of IF-ES cells in chimeric adults led to highly penetrant tumor formation by 12 months of age, causing multiple cancer types derived from the IF cells. Taken together, these data are consistent with global LOI having a causal role in tumorigenesis by affecting the regulation of the p53-p19Ar pathway and predisposing IF cells to transformation.by Teresa M. Holm.Ph.D

Global loss of imprinting leads to widespread tumorigenesis in adult mice

SummaryLoss of imprinting (LOI), commonly observed in human tumors, refers to loss of monoallelic gene regulation normally conferred by parent-of-origin-specific DNA methylation. To test the function of LOI in tumorigenesis, we developed a model by using transient demethylation to generate imprint-free mouse embryonic stem cells (IF-ES cells). Embryonic fibroblasts derived from IF-ES cells (IF-MEFs) display TGFβ resistance and reduced p19 and p53 expression and form tumors in SCID mice. IF-MEFs exhibit spontaneous immortalization and cooperate with H-Ras in cellular transformation. Chimeric animals derived from IF-ES cells develop multiple tumors arising from the injected IF-ES cells within 12 months. These data demonstrate that LOI alone can predispose cells to tumorigenesis and identify a pathway through which immortality conferred by LOI lowers the threshold for transformation

Probucol Prevents Early Coronary Heart Disease and Death in the High-Density Lipoprotein Receptor SR-BI/Apolipoprotein E Double Knockout Mouse

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5-6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between approximately 3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease

To Transect or Not Transect : Results from the Scandinavian Urethroplasty Study, A Multicentre Randomised Study of Bulbar Urethroplasty Comparing Excision and Primary Anastomosis Versus Buccal Mucosal Grafting

Background: Open surgical treatment of short bulbar urethral strictures (urethroplasty) is commonly performed as transecting excision and primary anastomosis (tEPA) or buccal mucosa grafting (BMG). Erectile dysfunction and penile complications have been reported, but there is an absence of randomised trials. Objective: To evaluate sexual dysfunction and penile complications after urethroplasty with tEPA versus BMG. Design, setting, and participants: Centres in Finland, Sweden and Norway participated. Patients with a bulbar urethral stricture of Intervention: Patients were randomised to either tEPA or BMG urethroplasty. Outcome measurements and statistical analysis: Sexual dysfunction was measured using the International Index of Erectile Function, 5-item version (IIEF-5) and a penile complications questionnaire (PCQ) designed for this study. Continuous data were analysed using analysis of covariance and categorical data were compared using a chi(2) test. Results and limitations: A total of 151 patients were randomised to either tEPA (n = 75) or BMG (n = 76). The tEPA group reported more penile complications (p = 0.02), especially reduced glans filling (p = 0.03) and a shortened penis (p = 0.001). There were no differences in postoperative IIEF-5 total scores. Recurrence rates were similar in both groups (12.9%) but the study was not designed to detect differences in recurrence rates. The PCQ is not validated, which is a limitation. Conclusions: More patients reported penile complications after urethroplasty with tEPA than with BMG. This should be considered when choosing the operative method, and patients should be informed accordingly. Patient summary: This study compared two common operations for repair of narrowing of the male urethra. Neither of the two methods seems to cause worsened erections. However, penile problems are more common after the transection technique than after the grafting technique. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association of Urology.Peer reviewe

Investigating Cellular Structures at the Nanoscale with Organic Fluorophores

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging

Economic evaluation of timber and non-timber forest products of in Yucatan State, Mexico: A case study of Cordia dodecandra (Ciricote) tree

152 p.This study is concerned with comparisons between timber and fruit production from Ciricote tree (Cordia dodecandra) in agroforestry scheme in Yucatán Península, México. The Mexican Department of Forestry (CONAFOR) is now encouraging research programs on reforestation with native tree species in order to increase farmers´ income and reduce deforestation and soil degradation. One eligible species for reforestation is the multi-purpose tree Ciricote. In order to assess the market potential and to compare the economic benefits of fruits and timber for rural households, an economic evaluation was undertaken from the household level up to the local first-level of buyers. The methodology used for this purpose was the Cost-Benefit-Analysis (CBA), using for decision criterias: Net Present Value (NPV), Internal Rate of Return (IRR) and Benefit Cost Rate (BCR). The financial analysis for a 25 and 40 years rotation period shows that timber production presents higher net present value and benefit cost rate than fruit production. Only for a 60 year period and a discount rate higher than 9%, fruit production presents a higher NPV than timber production. With regard to the value chains for timber and fruit production great differences can be observed. The timber value chain has more actors involved than the fruit production value chain, therefore the transaction costs in lumber industry are higher. Nevertheless timber has higher market than fruit. Approximately 1000 m3 of timber are demanded by the market which means that there is a potential demand of 300 has of ciricote tree. The price of ciricote timber could be reached 45 pesos/board feet vs 30 pesos/board feet of mahogany (Swietenia macrophylla). The price of ciricote fruit is 1,5 pesos/kilo and the potential market for fruit production (22 900 kg of raw fruits) will be covered with 6.0 – 7.0 ha ha of ciricote for fruit production. It was found that the additional income from other sources is very important for maya peasants due to participation in farm as well as non-farm activities. Farm households diversify their economic activities in order to cope with crop shortfalls and to overcome credit and land market constraints. Ciricote based agroforestry systems contribute to income and risk minimization by diversification of crops

Developing non-formal learning activities focused on increasing evolutionary knowledge and scientific literacy

Non-formal learning activities have an important role in continuous education of the general audience, and thus provide an essential element in increasing evolutionary knowledge and scientific literacy for European citizens, beyond the formal schooling. This guide brings together explanations of how non-formal learning can be designed, with special emphasis on evaluating the activities. The guide describes common activity types and examples encountered in our scoping conducted via surveys, personal contacts, and literature and internet search. It also provides reflection points for practitioners (activity designers, educators, science communicators) to embed evaluation as a form of engaging activity participants, as well as a tool to assess the impact and improve practice.info:eu-repo/semantics/publishedVersio

Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies

Phytoplankton dynamics in relation to seasonal variability and upwelling and relaxation patterns at the mouth of Ria de Aveiro (West Iberian Margin) over a four-year period

From June 2004 to December 2007, samples were weekly collected at a fixed station located at the mouth of Ria de Aveiro (West Iberian Margin). We examined the seasonal and inter-annual fluctuations in composition and community structure of the phytoplankton in relation to the main environmental drivers and assessed the influence of the oceano-graphic regime, namely changes in frequency and intensity of upwelling events, over the dynamics of the phytoplankton assemblage. The samples were consistently handled and a final subset of 136 OTUs (taxa with relative abundance > 0.01%) was subsequently submitted to various multivariate analyses. The phytoplankton assemblage showed significant changes at all temporal scales but with an overriding importance of seasonality over longer-(inter-annual) or shorter-term fluctuations (upwelling-related). Sea-surface temperature, salinity and maximum upwelling index were retrieved as the main driver of seasonal change. Seasonal signal was most evident in the fluctuations of chlorophyll a concentration and in the high turnover from the winter to spring phytoplankton assemblage. The seasonal cycle of production and succession was disturbed by upwelling events known to disrupt thermal stratification and induce changes in the phytoplankton assemblage. Our results indicate that both the frequency and intensity of physical forcing were important drivers of such variability, but the outcome in terms of species composition was highly dependent on the available local pool of species and the timing of those events in relation to the seasonal cycle. We conclude that duration, frequency and intensity of upwelling events, which vary seasonally and inter-annually, are paramount for maintaining long-term phytoplankton diversity likely by allowing unstable coexistence and incorporating species turnover at different scales. Our results contribute to the understanding of the complex mechanisms of coastal phytoplankton dynamics in relation to changing physical forcing which is fundamental to improve predictability of future prospects under climate change.Portuguese Foundation for Science and Technology (FCT, Portugal) [SFRH/BPD/ 94562/2013]; FEDER funds; national funds; CESAM [UID/AMB/50017]; FCT/MEC through national funds; FEDERinfo:eu-repo/semantics/publishedVersio