Telomeres, Sex and Epigenetics: The role of mouse HP1γ on telomere stability and sexual dimorphism

Heterochromatin Protein 1 (HP1) is a protein family of epigenetic modifiers that are integral units of heterochromatin establishment with three mammalian isoforms HP1α, HP1β and HP1γ. All three isoforms are found in heterochromatic regions where they assist with chromatin compaction and gene silencing, yet, HP1γ is also enriched on euchromatic regions with a suggestive gene activating role. In mice, HP1γ has been shown to influence genes that differ between the sexes, nonetheless, the implications of this sexual dimorphism and the molecular mechanisms underlying it are poorly understood. Here, I show that HP1γ is important for male cell proliferation and its absence causes earlier onset of cellular senescence in both sexes. The effect of HP1γ loss is further reflected in male embryo growth rate. Cleavage under targets and release using nuclease (CUT & RUN) analysis revealed that HP1γ is binding on genes differentially expressed among the sexes affecting their expression. Preliminary data using Super-Low Input Carrier Cap analysis of gene expression (SLIC-CAGE) suggests that HP1γ most likely does not regulate cryptic transcription in mice. HP1γ was also enriched on repetitive DNA sequences at the end of chromosomes termed telomeres. Given the recruitment of HP1γ at telomeres for heterochromatin formation and the direct link between senescence and telomere length, we examined the role of this factor on telomere maintenance. Loss of mouse HP1γ leads to a downregulation of various telomere and telomere-accessory transcripts, including shelterin protein TRF1. This transcriptional and protein downregulation is associated with increased telomere replication stress and DNA damage, both effects more profound in females. My analysis suggests that the source of the impaired telomere replication is the increase in telomeric DNA:RNA hybrids due to the upregulation of TElomeric Repeatcontaining RNA (TERRA) arising from mouse chromosome 18 and chromosome X. Overall, this PhD thesis showcases the important role of HP1γ on sexual dimorphism and telomere stability during early mouse development.Open Acces

Braid groups, mapping class groups, and Torelli groups

This thesis discusses subgroups of mapping class groups of particular surfaces. First, we study the Torelli group, that is, the subgroup of the mapping class group that acts trivially on the first homology. We investigate generators of the Torelli group, and we give an algorithm that factorizes elements of the Torelli group into products of particular generators. Furthermore, we investigate normal closures of powers of standard generators of the mapping class group of a punctured sphere. By using the Jones representation, we prove that in most cases these normal closures have infinite index in the mapping class group. We prove a similar result for the hyperelliptic mapping class group, that is, the group that consists of mapping classes that commute with a fixed hyperelliptic involution. As a corollary, we recover an older theorem of Coxeter (with 2 exceptional cases), which states that the normal closure of the m-th power of standard generators of the braid group has infinite index in the braid group. Finally, we study finite index subgroups of braid groups, namely, congruence subgroups of braid groups. We discuss presentations of these groups and we provide a topological interpretation of their generating sets

The effect of chronic stress on the adolescent brain, learning, and memory

Adolescence is a period in which major psychological and neural changes occur, along with changes in the stress response. This has resulted in some researchers characterising this developmental period as one of “storm and stress”. Interestingly, the brain regions implicated in learning and memory processes are both particularly sensitive to stress and undergoing major development during adolescence. Therefore, the broad aim of this thesis was to examine the impact of chronic stress exposure early in life on learning and memory processes that underlie a number of mental disorders. In the first two empirical Chapters, I examined pharmacological and endogenous activation of Tropomyosin receptor kinase B (TrkB), a key mediator of activity-dependent plasticity underlying memory, during Pavlovian fear conditioning and extinction in adolescent rats. In Chapter 2 I examined the effects of an agonist of TrkB on extinction retention in both non-stressed adolescent rodents and those that had been exposed to chronic levels of corticosterone, a stress-related hormone. Pharmacological activation of TrkB during extinction training facilitated extinction retention in non-stressed adolescent rats, but not in those exposed to chronic corticosterone. In Chapter 3 I examined levels of full-length, truncated, and activated (phosphorylated) TrkB receptors in three key brain regions involved in extinction: the ventral hippocampus, prefrontal cortex, and basolateral amygdala, of adult and adolescent rats following extinction training. One pertinent finding of this work was a significantly higher ratio of truncated to full-length TrkB receptors in the ventral hippocampus of adults than adolescents. In Chapter 4 human participants (older adolescents and adults) were exposed to an acute stressor (the cold pressor test) prior to viewing trauma images and provided salivary samples for analyses of the stress hormone cortisol. Participants recorded instances of intrusive, involuntary memories of these images over subsequent days. No age differences were detected in relation to the number of intrusive memories reported by participants, or their cortisol response to the cold pressor test. However, individuals with higher levels of early-life adversity were significantly more affected by exposure to trauma images. Taken together, these experiments extend knowledge of the impact of chronic stress and adversity in adolescence on learning and memory

Chapter Solution-Processed Graphene-Based Transparent Conductive Electrodes as Ideal ITO Alternatives for Organic Solar Cells

The isolation of free-standing graphene in 2004 was the spark for a new scientific revolution in the field of optoelectronics. Due to its extraordinary optoelectronic and mechanical properties, graphene is the next wonder material that could act as an ideal low-cost alternative material for the effective replacement of the expensive conventional materials used in organic optoelectronic applications. Indeed, the enhanced electrical conductivity of graphene combined with its high transparency in visible and near-infrared spectra, enabled graphene to be an ideal low-cost indium tin oxide (ITO) alternative in organic solar cells (OSCs). The prospects and future research trend in graphene-based TCE are also discussed. On the other hand, solution-processed graphene combines the unique optoelectrical properties of graphene with large area deposition and flexible substrates making it compatible with printing and coating technologies, such as roll-to-roll, inkjet, gravure, and flexographic printing manufacturing methods. This chapter provides an overview of the most recent research progress in the application of solution-processed graphene-based films as transparent conductive electrodes (TCEs) in OSCs. (a) Chemically converted graphene (CCG), (b) thermally and photochemically reduced graphene oxide, (c) composite reduced graphene oxide-carbon nanotubes, and (d) reduced graphene oxide mesh films have demonstrated their applicability in OSCs as transparent, conductive electrodes

Solution-Processed Graphene-Based Transparent Conductive Electrodes as Ideal ITO Alternatives for Organic Solar Cells

The isolation of free-standing graphene in 2004 was the spark for a new scientific revolution in the field of optoelectronics. Due to its extraordinary optoelectronic and mechanical properties, graphene is the next wonder material that could act as an ideal low-cost alternative material for the effective replacement of the expensive conventional materials used in organic optoelectronic applications. Indeed, the enhanced electrical conductivity of graphene combined with its high transparency in visible and near-infrared spectra, enabled graphene to be an ideal low-cost indium tin oxide (ITO) alternative in organic solar cells (OSCs). The prospects and future research trend in graphene-based TCE are also discussed. On the other hand, solution-processed graphene combines the unique optoelectrical properties of graphene with large area deposition and flexible substrates making it compatible with printing and coating technologies, such as roll-to-roll, inkjet, gravure, and flexographic printing manufacturing methods. This chapter provides an overview of the most recent research progress in the application of solution-processed graphene-based films as transparent conductive electrodes (TCEs) in OSCs. (a) Chemically converted graphene (CCG), (b) thermally and photochemically reduced graphene oxide, (c) composite reduced graphene oxide-carbon nanotubes, and (d) reduced graphene oxide mesh films have demonstrated their applicability in OSCs as transparent, conductive electrodes

A Hybrid Genetic Programming Method in Optimization and Forecasting: A Case Study of the Broadband Penetration in OECD Countries

The introduction of a hybrid genetic programming method (hGP) in fitting and forecasting of the broadband penetration data is proposed. The hGP uses some well-known diffusion models, such as those of Gompertz, Logistic, and Bass, in the initial population of the solutions in order to accelerate the algorithm. The produced solutions models of the hGP are used in fitting and forecasting the adoption of broadband penetration. We investigate the fitting performance of the hGP, and we use the hGP to forecast the broadband penetration in OECD (Organisation for Economic Co-operation and Development) countries. The results of the optimized diffusion models are compared to those of the hGP-generated models. The comparison indicates that the hGP manages to generate solutions with high-performance statistical indicators. The hGP cooperates with the existing diffusion models, thus allowing multiple approaches to forecasting. The modified algorithm is implemented in the Python programming language, which is fast in execution time, compact, and user friendly

Mechanistic studies of gold-catalyzed reactions between aromatic nitrogen heterocycles and alkynes using Density Functional Theory calculations.

Στην παρούσα διδακτορική διατριβή επιχειρείται η μελέτη του μηχανισμού αντιδράσεων καταλυόμενων από καταλύτες χρυσού με τη χρήση υπολογισμών DFT (Θεωρίας Πυκνότητας Συναρτησιοειδούς). Mελετήσαμε τις καταλυόμενες από χρυσό (Ι) αντιδράσεις 1) [4+2] προσθήκης των 1,3-διινίων με δακτυλίους πυρολίου και ινδολίου που οδηγούν σε υποκατεστημένα ινδόλια και καρβαζόλια αντίστοιχα 2) την [3+2] προσθήκη αλκινίων στο ανθρανύλιο που οδηγούν στον σχηματισμό 7-ακυλο-ινδολίων και 3) την [3+2] προσθήκη των ανθρανυλίων, των 1,2,4-οξαδιαζολίων ή των 4,5-διυδρο-1,2,4-οξαδιαζολίων και του ιναμιδίου PhC≡C-N(Ts)Me, που παράγει τόπο-εκλεκτικά 2-αμινο-3-φαίνυλο-7-ακυλο ινδόλια, Ν-ακυλο-5-αμινο-ιμιδαζόλια ή Ν-άλκυλο-4-αμινο-ιμιδαζόλια, αντίστοιχα. Στην αντίδραση 1, τα μονοπάτια της αντίδρασης βρέθηκαν περισσότερο περίπλοκα από ότι είχαμε υποθέσει. Στην περίπτωση του σχηματισμού των καρβαζολίων, ένα ανταγωνιστικό μονοπάτι περιλαμβάνει τον σχηματισμό ενός απρόσμενου σπειρανικού ενδιαμέσου που προχωρά μέσω μετάθεσης 1,2-αλκινίου. Η παρούσα μελέτη εξυπηρετεί στο να δώσει έμφαση στη σημασία που έχουν τα στοιχειώδη βήματα ενός μηχανισμού, που συχνά παραβλέπονται ως άσχετα με την κινητική, όπως η απόσπαση πρωτονίου/κατιόντος χρυσού από τα ενδιάμεσα τα αντίδρασης. Στις αντιδράσεις 2 και 3 η παρατηρούμενη τόπο- εκλεκτικότητα φαίνεται να συνδέεται με τον μη αντιστρεπτό σχηματισμό του κομβικής σημασίας ενδιαμέσου α-ιμινο-χρυσού καρβενίου, μέσω της αρχικής τόπο- εκλεκτικής πυρηνόφιλης προσβολής του ατόμου του Ν του ανθρανυλίου στο τμήμα του αλκινίου. Η ακόλουθη απότομη πτώση της ενέργειας σχηματίζει το κομβικής σημασίας καρβένιο του α-ιμινο-χρυσού. Στην περίπτωση του 1,2,4-οξαδιαζόλιου ή του 4,5- διύδρο-1,2,4-οξαδιαζόλιο, αντί για το σχηματισμό ενός πενταμελούς δακτυλίου από το καρβένιο του α-ιμινο-χρυσού, βρέθηκε ένα ανταγωνιστικό μονοπάτι που περιλαμβάνει τον σχηματισμό ενδιαμέσων αποτελούμενων από ένα τετραμελή δακτύλιο συμπυκνωμένο με έναν τριμελή, το οποίο μετά από μετάθεση και διάνοιξη του δακτυλίου οδήγησε στον ιμιδαζολικό δακτύλιο.In this thesis we studied the mechanism of series of gold catalyzed reactions with the use of DFT (Density Functional Theory) computations. We studied the gold(I) catalyzed reaction of 1) [4+2] addition reaction of 1,3-diynes with pyrrole and indole rings leading to substituted indoles and carbazoles, respectively 2) [3+2] addition reaction of alkynes onto anthranils that furnishes 7-acylindoles and 3) [3+2] reaction between anthranil, 1,2,4-oxadiazole or 4,5-dihydro-1,2,4-oxadiazole and an ynamide, PhC≡C-N(Ts)Me that produces regioselectively 2-amino-3-phenyl-7-acyl indoles, N-acyl-5-aminoimidazoles or N-alkyl-4-aminoimidazoles, respectively. For the reaction 1, pathways are found to be significantly more complex than we had anticipated. In the case of carbazoles formation, one competitive route involves the generation of an unexpected spirane intermediate evolving via a 1,2-alkenyl migration. Τhe current study also serves to highlight the importance of fundamental steps that are often disregarded as kinetically irrelevant, such as the protodeauration of reaction intermediates. For the reactions 2 and 3, the observed regioselectivity seems to be connected to the irreversible formation of a key α-imino gold carbene intermediate, common to all reaction profiles, through the initial regioselective nucleophilic attack of the anthranil N atom onto the alkyne fragment. A subsequent and steep energy drop furnishes the key α-imino gold carbene. In the case of 1,2,4-oxadiazoles or 4,5-dihydro-1,2,4-oxadiazoles are found, instead of the formation of a five-member ring from the α-imino gold carbene, one competitive route involves the formation of intermediates consisting of a four-member ring condensed with a three-member ring which after a metathesis and ring expansion led to the imidazole ring

On the mechanism of the Au(I)‐mediated addition of alkynes to anthranils to furnish 7‐acylindoles

Indole is a very common structural motif in alkaloids with a remarkable history in pharma industry. In the continuous search for more direct and efficient access to these valuable structures, a new and rather elegant approach was found by Jin and coworkers, which involved a gold(I)-mediated addition of alkynes onto anthralins. This approach selectively furnishes 7-acylindoles in a rather expeditious way, and it has been shown to be compatible with a large range of decorated reactants, both at the alkyne side and at the anthralin side. We studied the mechanism of this reaction with a set of different alkynes, including disubstituted ones, to establish similarities and differences between them and to aid in the elucidation of key steps in the reaction pathway. The observed regioselectivity seems to be connected to the irreversible formation of a key α-imino gold carbene intermediate, common to all reaction profiles, through the initial regioselective nucleophilic attack of the anthranil N atom onto the alkyne fragment.Xunta de Galicia | Ref. ED431C 2021/41Agencia Estatal de Investigación | Ref. PID2020‐115789GB‐C22Financiado para publicación en acceso aberto: Universidade de Vigo/CISU

CRISPR-mediated reactivation of DKK3 expression attenuates TGF-beta signaling in prostate cancer

The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-β/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the DKK3 promoter, induces DKK3 expression, and inhibits TGF-β/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-β/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-β. TGF-β signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-β found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-β signaling

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-β Signaling in Prostate Cancer

The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-β/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the DKK3 promoter, induces DKK3 expression, and inhibits TGF-β/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-β/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-β. TGF-β signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-β found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-β signaling