Search

BACKGROUND: A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs). METHODS: We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing. RESULTS: We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC. CONCLUSIONS: Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

The cell is the smallest unit of life and contains DNA, RNA, proteins and a variety of other macromolecules. In recent years, technological advances in the field of single cell biology have revealed a staggering amount of phenotypic heterogeneity between cells in a population, which were previously considered homogenous. Previous work has largely been focused on studies of RNA. As proteins however are the ultimate effectors of genetic information, this thesis aims to provide a protein-centered view on cellular heterogeneity, particularly focusing on cell cycle and cellular metabolism. Most of my work has been performed within the framework of the Human Protein Atlas project. In the context of this project, we mapped the spatial distribution of more than 13.000 human proteins with subcellular resolution and found that around a quarter of all human proteins exhibit protein expression heterogeneity. In Paper I, we hypothesized that a majority of the observed cellular heterogeneity can be explained by differences in cell cycle progression. Therefore, we generated a map of proteomic and transcriptomic heterogeneity at subcellular resolution, which we precisely aligned to the cell cycle position of individual cells. This approach allowed us to identify hundreds of previously unknown cell cycle-related proteins. With sustained proliferative signaling representing a hallmark of cancer, novel cell-cycle proteins could serve as potential new drug targets against cancer. We further show that a large part of cell cycle dependent proteome variability is not established by transcriptomic cycling. This suggests that post-translational modifications are a major contributor to the regulation of cell cycle dependent protein level changes. Therefore, in Paper II, we carried out a deep phosphoproteome mass spectrometry profiling of the same cellular model as in Paper I and identified almost 5,000 cell cycle dependent phosphosites on over 2,000 proteins. The unprecedented scale of our p, Cellen är den minsta enheten av liv, och varje cell innehåller en komplex uppsättning av DNA, RNA, proteiner och andra makromolekyler. Under de senaste åren har teknologiska framsteg inom cellbiologiska studier av enskilda celler avslöjat en överväldigande mängd fenotypisk heterogenitet mellan cellpopulationer som tidigare betraktades som homogena. Tidigare kartläggning av denna heterogenitet har främst fokuserat på studier av RNA. Denna avhandling syftar dock till att ge en proteincentrerad syn på cellulär variabilitet, med särskilt fokus på cellcykeln och cellulär metabolism. Det mesta av mitt arbete innefattar data från Human Protein Atlas-projektet. I detta projekt har vi kartlagt den spatiala fördelningen av över 13 000 mänskliga proteiner med subcellulär upplösning. Vi finner att ungefär en fjärdedel av alla mänskliga proteiner uppvisar heterogenitet i proteinuttryck mellan enskilda celler. I Artikel I undersökte vi hypotesen att majoriteten av den observerade cellulära heterogeniteten kan förklaras av skillnader i cellcykelprogression. Därför genererade vi en stor karta över proteomisk och transkriptomisk uttrycksdata i relation till den mänskliga cellcykeln i enskilda celler. Vi identifierade hundratals nya proteiner relaterade till cellcykeln, vilka kan komma att fungera som potentiella läkemedelsmål vid sjukdomar som cancer. Vi visar vidare att en stor del av den cellcykelberoende variabiliteten på proteinnivå inte etableras genom cykliska ändringar av transkriptomet. Detta antyder att posttranslationella modifieringar i betydande utsträckning bidrar till regleringen av dynamiska förändringar i proteinuttryck under cellcykeln. Därför utförde vi i Artikel II en djup masspektrometriprofilering av fosfoproteomet längs samma cellulära modell som i Artikel I och identifierade över 2000 cellcykelberoende fosforyleringsplatser hos människans proteiner. Den oöverträffade omfattningen av denna fosfoproteomiska data gör att vi kan koppla cellcykelberoende dynamik i, QC 2024-04-08

PhD thesis "To Rebehold the Cell: Natural and Synthetic Cell Cycles Through the Lenses of Macromolecular Motion"; Chapter 5 "A synthetic, homeostatic genetic circuit enables titration-based control of DNA replication initiation in Escherichia coli"; Supplementary Material. Contains supplementary tables, figures and the source data used to generate the figures in the main text.

Background: Receptor-type tyrosine-protein phosphatase T (PTPRT) is a transmembrane protein that is involved in cell adhesion. We previously found that PTPRT was downregulated in multiple cancer types and the mutation of PTPRT was associated with cancer early metastasis. However, the impacts of PTPRT downregulation on tumour proliferation, invasion, and clinical interventions such as immune checkpoint inhibitor (ICI) therapies remained largely unknown. Methods: Gene expression data of non-small cell lung cancer (NSCLC) samples from The Cancer Genome Atlas database were downloaded and used to detect the differential expressed genes between PTPRT-high and PTPRT-low subgroups. Knockdown and overexpress of PTPRT in lung cancer cell lines were performed to explore the function of PTPRT in vitro. Western blot and qRT-PCR were used to evaluate the expression of cell cycle-related genes. CCK-8 assays, wound-healing migration assay, transwell assay, and colony formation assay were performed to determine the functional impacts of PTPRT on cell proliferation, migration, and invasion. KM-plotter was used to explore the significance of selected genes on patient prognosis. Results: PTPRT was found to be downregulated in tumours and lung cancer cell lines compared to normal samples. Cell cycle-related genes (BIRC5, OIP5, and CDCA3, etc.) were specifically upregulated in PTPRT-low lung adenocarcinoma (LUAD). Modulation of PTPRT expression in LUAD cell lines affected the expression of BIRC5 (survivin) significantly, as well as the proliferation, migration, and invasion of tumour cells. In addition, low PTPRT expression level was correlated with worse prognosis of lung cancer and several other cancer types. Furthermore, PTPRT downregulation was associated with elevated tumour mutation burden and tumour neoantigen burden in lung cancer, indicating the potential influence on tumour immunogenicity. Conclusion: Our findings uncovered the essential roles of PTPRT in the regulation of prol

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in reg- ulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk62/2 HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more sus- ceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcrip- tional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABLp2101 LSCs. Transplantation with BCR- ABLp2101–infected bone marrow from Cdk62/2 mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk62/2 BCR-ABLp2101 LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

Work in the authors’ laboratories is funded by grants from MINECO (SAF2012-36079 to PB and SAF201238215, SAF2014-57791-REDC, and BFU2014-52125-REDT to MM), and Comunidad de Madrid (S2010/BMD2470)., An attractive strategy for cancer therapy is to stop cell proliferation by means of agents that directly arrest the cell cycle. Microtubule poisons such as taxanes block mitosis, eventually leading to cell death in a process frequently known as mitotic catastrophe. However, some cells are able to bypass this mitotic arrest and survive, thus contributing to chemoresistance to those therapies. We have recently observed that mitotic arrest induces an early autophagic flux response that results in autophagy-dependent mitochondrial degradation and a dramatic energetic deficit. The subsequent increase in the AMP/ATP ratio results in the activation of the metabolic sensor AMPK followed by phosphorylation and activation of PFKFB3, an enzyme required for glycolysis. Thus, mitophagy can be considered as a critical effector of the therapeutic effect of mitotic therapies, while both AMPK and PFKFB3 are critical for survival. The manipulation of these molecular routes may therefore have therapeutic benefits in the presence of microtubule poisons., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

Salazar-Roa, Maria as first author. M.S.R. thanks the Asociación Española contra el Cáncer (AECC) for support. Research in the laboratory of the authors is supported by grants from the Comunidad de Madrid (S2010/BMD-2470), Spanish Ministry of Economy and Competitiveness (BFU2014-52125-REDT, SAF2014-57791-REDC, PCIN-2015-007 and SAF2015-69920-R cofunded by ERDF-EU), Worldwide Cancer Research (WCR no. 15-0278), and the European Commission Seventh Framework Programme (MitoSys project HEALTH-F5-2010-241548 and NEURON8-Full-815-094)., Cell division is a complex process with high energy demands. However, how cells regulate the generation of energy required for DNA synthesis and chromosome segregation is not well understood. Recent data suggest that changes in mitochondrial dynamics and metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis crosstalk with, and are tightly regulated by, the cell division machinery. Alterations in energy availability trigger cell-cycle checkpoints, suggesting a bidirectional connection between cell division and general metabolism. Some of these connections are altered in human disease, and their manipulation may help in designing therapeutic strategies for specific diseases including cancer. We review here recent studies describing the control of metabolism by the cell-cycle machinery., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

E.D., C.M. and M.S.-R. were supported by the Spanish Fondo de Investigaciones Sanitarias (Madrid), MINECO (Juan de la Cierva programme) and Asociación Española contra el Cáncer (AECC), respectively. L.E.-M. is a recipient of a JAE predoctoral fellowship from the CSIC. A.K.S. was supported by USPHS grants RO1DK19514, RO1DK67509. G.V. was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) and Fondo Europeo de Desarrollo Regional (FEDER) (PI12/02248), Fundació La Marató de TV3 (m12 20134031), and Fundación Mutua Madrileña (AP101042012). M.L. was supported by the European Community’s Seventh Framework Programme under grant agreement no. 281854—the ObERStress (European Research Council project). E.R. was financially supported by a MINECO grant (SAF 2010-20256). Work in the R.M. laboratory was supported by the Fundación Botín, Banco Santander and MINECO (BFU2011-30121, BFU2014-52125-REDT and Consolider RNAREG CSD2009-00080). Work in the P.B. laboratory is supported by a grant from the Spanish Ministry for Economy and Competitiveness (MINECO; SAF2012-36079). Work in the M.M. laboratory was supported by grants from the MINECO (SAF2012-38215), Consolider-Ingenio 2010 Programme (SAF2014-57791-REDC), Excellence Network CellSYS (BFU2014-52125-REDT), the OncoCycle Programme (S2010/BMD-2470) from the Comunidad de Madrid, Worldwide Cancer Research (WCR no. 15-0278), and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548)., Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

The development of CRISPR-derived genome editing technologies enables precise manipulation of DNA sequences within the human genome. One class of these editing tools is base editors, which install point mutations with high efficiency and specificity, without the disadvantages of traditional Cas9-based nucleases. This is due to the introduction of uracil and inosine intermediates, as opposed to breakage of the DNA backbone. As such, they rely on more ubiquitous DNA repair pathways than wild type Cas9. Cas9 is also highly reliant on cell cycle phase, depending on homology-directed repair (HDR), which is only active in late S and G2 phases. To date, no one has investigated the relationship of base editors to the cell cycle. We examine how reliant base editors are on the cell cycle using chemical synchronization experiments. We optimize protocols to evaluate base editing under synchronization conditions. We find that nickase-derived BEs function independently of the cell cycle, while non-nicking variants are highly dependent on S-phase processes. We discover that G1 synchronization during cytosine base editing causes significant increases in C•G to A•T “byproduct” introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We investigate the cause for C•G to A•T editing events and design an integrated fluorescent reporter system to identify genes implicated in these editing outcomes. We discover TLS polymerases POLI and POLK are likely responsible for these unique editing events. We further examine base editors’ response to nucleotide concentration to identify if that alters editing outcome. Finally, we develop and test a cell-cycle regulated base editor to apply our discovery towards technology development.

Summary Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and post-embryonic development of various organisms. Full activation of CDKs requires not only binding to cyclins but also phosphorylation of the T-loop domain. This phosphorylation is catalysed by CDK-activating kinases (CAKs). Plants have two distinct types of CAKs, namely CDKD and CDKF; in Arabidopsis, CDKF;1 exhibits the highest CDK kinase activity in vitro. We have previously shown that CDKF;1 also functions in the activation of CDKD;2 and CDKD;3 by T-loop phosphorylation. Here, we isolated the knockout mutants of CDKF;1 and showed that they had severe defects in cell division, cell elongation and endoreduplication. No defect was observed during embryogenesis, suggesting that CDKF;1 function is primarily required for post-embryonic development. In the cdkf;1 mutants, T-loop phosphorylation of CDKA;1, an orthologue of yeast Cdc2/Cdc28p, was comparable to that in wild-type plants, and its kinase activity did not decrease. In contrast, the protein level and kinase activity of CDKD;2 were significantly reduced in the mutants. Substitution of threonine-168 with a non-phosphorylatable alanine residue made CDKD;2 unstable in Arabidopsis tissues. These results indicate that CDKF;1 is dispensable for CDKA;1 activation but is essential for maintaining a steady-state level of CDKD;2, thereby suggesting the quantitative regulation of a vertebrate-type CAK in a plant-specific manner.

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T‐loop is required. This phosphorylation is catalyzed by CDK‐activating kinases (CAKs). In Arabidopsis three D‐type CDK genes (CDKD;1–CDKD;3) encode vertebrate‐type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy‐terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP‐binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK – CDKF;1 – exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T‐loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

A-type cyclins (CYCAs) are a type of mitotic cyclin and are closely related to cyclin B. Plant CYCAs are classified into three subtypes (CYCA1–CYCA3), among which CYCA3 has been suggested to show a biased expression during the G1-to-S phase. We characterised Arabidopsis CYCA3s (CYCA3;1–CYCA3;4) in terms of expression pattern and protein function. CYCA3;1 and CYCA3;2 transcripts were highly accumulated at the G1/S phase, whereas CYCA3;4 was constantly expressed during the cell cycle. Expressions of CYCA3;1 and CYCA3;2 were observed in actively dividing tissues, such as root and shoot apical meristems and lateral root primordia. Overexpression of CYCA3;1 or CYCA3;2 distorted apical dominance in Arabidopsis, indicating that they have critical functions in shoot meristems. In insect cells, CYCA3;1 formed an active kinase complex with CDKA;1, an orthologue of the yeast Cdc2/Cdc28p, and phosphorylated retinoblastoma-related protein, a key regulator in the transition from the G1 to the S phase. Our results suggest that Arabidopsis CYCA3;1 and CYCA3;2 are distinct members of the G1 cyclin family that play an important role in meristematic tissues.

The cell cycle is driven by the activity of cyclin-dependent kinase (CDK)–cyclin complexes. Therefore, internal and external signals converge on the regulation of CDK–cyclin activity to modulate cell proliferation in specific developmental processes and under various environmental conditions. CDK–cyclin activity is fine-tuned by multiple mechanisms, for example, transcriptional control, protein degradation, phosphorylation, and binding to CDK inhibitor. These molecular mechanisms underlie the regulation of the entry into or the exit from the cell cycle, the rate of cell cycle, or the transition from the mitotic cell cycle to the endocycle. The multiple mechanisms regulating CDK–cyclin activity coordinately enable the elaborate control of cell cycle by various upstream signals. Here, we review the molecular mechanisms that regulate the cell cycle and the endocycle in plants. We also introduce the recent progress in elucidating the regulatory mechanisms underlying plant development and the stress response in terms of cell-cycle control.

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

Cellular responses to DNA damage constitute one of the most important fields in cancer biology. In this class we will analyze classical and recent papers from the primary research literature to gain a profound understand of cell cycle regulation and DNA damage checkpoints that act as powerful emergency brakes to prevent cancer. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

type:Article, Ubiquitin specific protease (USP) 2 is a multifunctional deubiquitinating enzyme. USP2 modulates cell cycle progression, and therefore carcinogenesis, via the deubiquitination of cyclins and Aurora-A. Other tumorigenic molecules, including epidermal growth factor and fatty acid synthase, are also targets for USP2. USP2 additionally prevents p53 signaling. On the other hand, USP2 functions as a key component of the CLOCK/BMAL1 complex and participates in rhythmic gene expression in the suprachiasmatic nucleus and liver. USP2 variants influence energy metabolism by controlling hepatic gluconeogenesis, hepatic cholesterol uptake, adipose tissue inflammation, and subsequent systemic insulin sensitivity. USP2 also has the potential to promote surface expression of ion channels in renal and intestinal epithelial cells. In addition to modifying the production of cytokines in immune cells, USP2 also modulates the signaling molecules that are involved in cytokine signaling in the target cells. Usp2 knockout mice exhibit changes in locomotion and male fertility, which suggest roles for USP2 in the central nervous system and male genital tract, respectively. In this review, we summarize the cellular events with USP2 contributions and list the signaling molecules that are upstream or downstream of USP2. Additionally, we describe phenotypic differences found in the in vitro and in vivo experimental models.

BRG1 is mutated in 10% of lung cancers, making this mutation clinically relevant. The downstream effects of BRG1 included significantly affecting the cell cycle control and chromosomal replication pathway. CDK18, a cyclin-dependent kinase, was determined to be the gene with significantly decreased expression (p

The therapeutic efficacy of metformin in prostate cancer (PCa) appears uncertain based on various clinical trials. Metformin treatment failure may be attributed to the high frequency of transcriptional dysregulation, which leads to drug resistance. However, the underlying mechanism is still unclear. In this study, we found evidences that metformin resistance in PCa cells may be linked to cell cycle reactivation. Super-enhancers (SEs), crucial regulatory elements, have been shown to be associated with drug resistance in various cancers. Our analysis of SEs in metformin-resistant (MetR) PCa cells revealed a correlation with Prostaglandin Reductase 1 (PTGR1) expression, which was identified as significantly increased in a cluster of cells with metformin resistance through single-cell transcriptome sequencing. Our functional experiments showed that PTGR1 overexpression accelerated cell cycle progression by promoting progression from the G0/G1 to the S and G2/M phases, resulting in reduced sensitivity to metformin. Additionally, we identified key transcription factors that significantly increase PTGR1 expression, such as SRF and RUNX3, providing potential new targets to address metformin resistance in PCa. In conclusion, our study sheds new light on the cellular mechanism underlying metformin resistance and the regulation of the SE-TFs-PTGR1 axis, offering potential avenues to enhance metformins therapeutic efficacy in PCa.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. While neuronal contribution to FXS has been extensively studied in both animal and human-based models of FXS, the roles of astrocytes, a type of glial cells in the brain, are largely unknown. Here, we generated a human-based FXS model via differentiation of astrocytes from human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) and characterized their development, function, and proteomic profiles. We identified shortened cell cycle, enhanced Ca2+ signaling, impaired sterol biosynthesis, and pervasive alterations in the proteome of FXS astrocytes. Our work identified astrocytic impairments that could contribute to the pathogenesis of FXS and highlight astrocytes as a novel therapeutic target for FXS treatment.

First described in the early 20th century, diurnal oscillations in stem cell proliferation exist in multiple internal epithelia, including in the gastrointestinal tract, and in the epidermis. In the mouse epidermis, 3- to 4-fold more stem cells are in S-phase during the night than during the day. More recent work showed that an intact circadian clock intrinsic to keratinocytes is required for these oscillations in epidermal stem cell proliferation. The circadian clock also regulates DNA excision repair and DNA damage in epidermal stem cells in response to ultraviolet B radiation. During skin inflammation, epidermal stem cell proliferation is increased and diurnal oscillations are suspended. Here we discuss possible reasons for the evolution of this stem cell phenomenon. We argue that the circadian clock coordinates intermediary metabolism and the cell cycle in epidermal stem cells to minimize the accumulation of DNA damage from metabolism-generated reactive oxygen species. Circadian disruption, common in modern society, leads to asynchrony between metabolism and the cell cycle, and we speculate this will lead to oxidative DNA damage, dysfunction of epidermal stem cells, and skin aging.

The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5-/- HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5-/- mice, suggesting that the Irx5-/- HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5-/-mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer.

Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely quantify cell-cycle effects and diverse cell states orchestrated by specific CDKs. While pairwise disruption of CDK4/6 is synthetic-lethal, only CDK6 is required for normal cell-cycle progression and transcriptional activation. Multiple CDKs (CDK1/7/9/12) are synthetic-lethal in combination with PRMT5, independent of cell-cycle control. In-depth analysis of mRNA expression and splicing patterns provides multiple lines of evidence that the CDK-PRMT5 dependency is due to aberrant transcriptional regulation resulting in premature termination. These inter-dependencies translate to drug-drug synergies, with therapeutic implications in cancer and other diseases.

Identifying effective therapeutic treatment strategies is a major challenge to improving outcomes for patients with breast cancer. To gain a comprehensive understanding of how clinically relevant anti-cancer agents modulate cell cycle progression, here we use genetically engineered breast cancer cell lines to track drug-induced changes in cell number and cell cycle phase to reveal drug-specific cell cycle effects that vary across time. We use a linear chain trick (LCT) computational model, which faithfully captures drug-induced dynamic responses, correctly infers drug effects, and reproduces influences on specific cell cycle phases. We use the LCT model to predict the effects of unseen drug combinations and confirm these in independent validation experiments. Our integrated experimental and modeling approach opens avenues to assess drug responses, predict effective drug combinations, and identify optimal drug sequencing strategies.

Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess as well as mild neuronal excitation, such as chemoconvulsant‐induced brief seizures. Because most studies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear. Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle progression of precursors was analysed by systemic administration of thymidine analogues. We found that brief seizures immediately resulted in cell cycle retardation in the SVZ. However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ was followed by enhanced cell cycle re‐entry after the first round of mitosis, leading to precursor pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient. Cell cycle progression in the PTZ‐treated group returned to normal after one cell cycle. The numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendants of SVZ precursors, were not significantly different from those in control mice more than 2 days after seizures. Because similar effects were observed following electroconvulsive seizures, these responses are likely to be general effects of brief seizures. These results suggest that neurogenesis in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the SGZ., source:This is the pre-peer reviewed version of the following article: Mori, T., Wakabayashi, T., Hirahara, Y., Takamori, Y., Koike, T., Kurokawa, K. and Yamada, H. (2012), Differential responses of endogenous adult mouse neural precursors to excess neuronal exc, source:https://onlinelibrary.wiley.com/doi/full/10.1111/j.1460-9568.2012.08244.x

The B-Myb oncogene is overexpressed and increasingly considered a biomarker for several human cancers. B-Myb is vital in activating G2/M phase genes in the cell cycle. B-Myb forms the activating Myb-MuvB (MMB) complex to overcome the suppression of cell-cycle dependent genes caused by the MuvB-containing DREAM complex during quiescence. B-Myb interacts with the MuvB protein complex at cell-cycle gene promoters; however, the mechanism by which B-Myb activates the G2/M phase genes still needs to be fully understood. This study focuses on understanding the molecular mechanisms through which B-Myb and oncogenic kinases regulate G2/M phase gene activation through phosphorylation and interactions with nucleosomes and co-activators.

The adult human body is made up of trillions of cells and yet began as just one. This could not be possible without careful coordination of cell growth and cell division. The molecular mechanisms that drive and control cell growth as well as its coordination with the cell cycle remain poorly understood. Cell size is the outcome of cell growth. Thus, a decoupling of growth and division would result in cells of increasingly heterogenous sizes. Interestingly, the severity of a cancer is often correlated with increasingly heterogenous size of cells within a tissue. Furthermore, genes that are thought to play important roles in cell growth are often dysregulated in a variety of cancers. This suggests that the misregulation of mechanisms that control cell growth and cell size plays a role in cancer cell development. How cell growth influences cell cycle progression to control cell size is perhaps best understood in budding yeast where key cell cycle transitions are delayed until sufficient growth has occurred. It had been long believed that the predominant cell size checkpoint in budding yeast occurred at cell cycle entry. Recent work, however, suggests that bud growth strongly influences cell cycle progression in mitosis. In this thesis, I present evidence to support a model in which cells influence the extent and duration of bud growth by controlling mitotic exit, a cell cycle transition that seizes bud growth and finalizes daughter cell size.

BRG1 is mutated in 10% of lung cancers, making this mutation clinically relevant. The downstream effects of BRG1 included significantly affecting the cell cycle control and chromosomal replication pathway. CDK18, a cyclin-dependent kinase, was determined to be the gene with significantly decreased expression (p

Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or AT

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID N-4501-2014 (Sandra Sánchez Salcedo) ORCID 0000-0002-1889-2057 (Sandra Sánchez Salcedo), Hypothesis: Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200ºC) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700ºC) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Experiments: Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages)have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. Findings: This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone., Ministerio de Ciencia e Innovacion (MICINN), Ministerio de Economia y Competitividad (MINECO), Agening Network of Excellence, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

BackgroundTherapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative G0 state, which is difficult to capture and whose mutational drivers remain largely unknown.ResultsWe develop methodology to robustly identify this state from transcriptomic signals and characterise its prevalence and genomic constraints in solid primary tumours. We show that G0 arrest preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process and validate the role of the centrosomal gene CEP89 as a modulator of proliferation and G0 arrest capacity. Lastly, we demonstrate that G0 arrest underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single-cell data.ConclusionsWe propose a G0 arrest transcriptional signature that is linked with therapeutic resistance and can be used to further study and clinically track this state.

In the vertebrate eye, Notch ligands, receptors, and ternary complex components determine the destiny of retinal progenitor cells in part by regulating Hes effector gene activity. There are multiple paralogues for nearly every node in this pathway, which results in numerous instances of redundancy and compensation during development. To dissect such complexity at the earliest stages of eye development, we used seven germline or conditional mutant mice and two spatiotemporally distinct Cre drivers. We perturbed the Notch ternary complex and multiple Hes genes to understand if Notch regulates optic stalk/nerve head development; and to test intracellular pathway components for their Notch-dependent versus -independent roles during retinal ganglion cell and cone photoreceptor competence and fate acquisition. We confirmed that disrupting Notch signaling universally blocks progenitor cell growth, but delineated specific pathway components that can act independently, such as sustained Hes1 expression in the optic stalk/nerve head. In retinal progenitor cells, we found that among the genes tested, they do not uniformly suppress retinal ganglion cell or cone differentiation; which is not due differences in developmental timing. We discovered that shifts in the earliest cell fates correlate with expression changes for the early photoreceptor factor Otx2, but not with Atoh7, a factor required for retinal ganglion cell formation. During photoreceptor genesis we also better defined multiple and simultaneous activities for Rbpj and Hes1 and identify redundant activities that occur downstream of Notch. Given its unique roles at the retina-optic stalk boundary and cone photoreceptor genesis, our data suggest Hes1 as a hub where Notch-dependent and -independent inputs converge.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

The mammalian cell cycle is governed by a network of cyclin/Cdk complexes which signal the progression into the successive phases of the cell division cycle. Once coupled to the circadian clock, this network produces oscillations with a 24 h period such that the progression into each phase of the cell cycle is synchronized to the day-night cycle. Here, we use a computational model for the circadian clock control of the cell cycle to investigate the entrainment in a population of cells characterized by some variability in the kinetic parameters. Our numerical simulations showed that successful entrainment and synchronization are only possible with a sufficient circadian amplitude and an autonomous period close to 24 h. Cellular heterogeneity, however, introduces some variability in the entrainment phase of the cells. Many cancer cells have a disrupted clock or compromised clock control. In these conditions, the cell cycle runs independently of the circadian clock, leading to a lack of synchronization of cancer cells. When the coupling is weak, entrainment is largely impacted, but cells maintain a tendency to divide at specific times of day. These differential entrainment features between healthy and cancer cells can be exploited to optimize the timing of anti-cancer drug administration in order to minimize their toxicity and to maximize their efficacy. We then used our model to simulate such chronotherapeutic treatments and to predict the optimal timing for anti-cancer drugs targeting specific phases of the cell cycle. Although qualitative, the model highlights the need to better characterize cellular heterogeneity and synchronization in cell populations as well as their consequences for circadian entrainment in order to design successful chronopharmacological protocols., SCOPUS: ar.j, info:eu-repo/semantics/published

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Hepatocelularni i kolorektalni karcinom se sve ćešće javlja i ima visoku stopu smrtnosti. Postojanje aktivnih mehanizama hemorezistencije otežava korisnost hemoterapije. Upravo je pojava hemorezistencije podstakla opsežna istraživanja za razvoj novih terapeutskih agenasa i strategija za lečenje ovih vrsta karcinoma. Prirodni proizvodi biljnog porekla (sekundarni metaboliti) su bogat izvor potencijalnih lekova. Zbog toga smo u istraživanju usmerili pažnju na etil-acetatni ekstrakt biljke Frangula alnus (FA) i njegovu dominantnu komponentu emodin (E) i ispitali njihovo antikancerski potencijal na ćelijama hepatocelularnog (HepG2) i kolorektalnog (HCT116) karcinoma i citotoksičnost na normalnim MRC -5 ćelijama. Pokazan je snažan antioksidativni potencijal ekstrakta (DPPH i TBA test). Citotoksičnost ispitana MTT testom pokazala je jaku toksičnost obe supstance na ćelije HepG2 i HCT116, ali bez uticaja na MRC-5 ćelije. Dalje je analiza ćelijskog ciklusa pokazala da su i FA i E izazvali zaustavljanje u G1 fazi i blago nakupljanje ćelija u G2/M fazi. Pored toga, obe supstance su uvele ćelije u apoptozu i nekrozu i uticale su na mitohondrijalni membranski potencijal. Dalje, izazvali su značajan genotoksični efekat u kometnom testu na svim ćelijskim linijama. Može se zaključiti da su FA i E dobri kandidati za nove antikancerogene agense prirodnog porekla. Međutim, neophodna su dodatna istraživanja, posebno kada je u pitanju citotoksična aktivnost i bezbednost primene ovih supstanci., Hepatocellular and colorectal carcinoma is swiftly increasing alongside a high mortality rate. The existence of active mechanisms of chemoresistance hampers the usefulness of chemotherapy. The emergence of chemoresistance encouraged extensive research to develop new therapeutic agents and strategies for the treatment of these types of cancer. Natural products of plant origin (secondary metabolites) have been the most successful source of potential drug leads. Therefore, we turned our attention to the Frangula alnus ethyl-acetate extract (FA) and its dominant constituent emodin (E) and explored them for their anticancer potential on hepatocellular (HepG2) and colorectal (HCT116) carcinoma cells, and cytotoxicity on normal MRC-5 cells. Strong antioxidant potential of the extract was demonstrated (DPPH and TBA test). Cytotoxicity investigated by the MTT assay showed strong cell toxicity of both substances on HepG2 and HCT116 cells, but without affecting MRC-5 cells. Next, the analysis of cell cycles exhibited that both FA and E induced arrest in the G1 phase and slight accumulation of cells in the G2/M phase. In addition, both substances introduced cells into apoptosis and necrosis and modulated mitochondrial membrane potential. Further on, they caused significant genotoxic effect in comet assay applied in all cell lines. It can be concluded that FA and E are good candidates for new anticancer agents of natural origin. However, additional studies are necessary, especially when it comes to the cytotoxic activity and safety of the application of these substances.

The cytoplasmic Ca2+ concentration and the activity of K+ channels on the plasma membrane regulate cellular processes ranging from mitosis to oriented migration. The interplay between Ca2+ and K+ signals is intricate, and different cell types rely on peculiar cellular mechanisms. Derangement of these mechanisms accompanies the neoplastic progression. The calcium signals modulated by voltage-gated (KV) and calcium-dependent (KCa) K+ channel activity regulate progression of the cell division cycle, the release of growth factors, apoptosis, cell motility and migration. Moreover, KV channels regulate the cell response to the local microenvironment by assembling with cell adhesion and growth factor receptors. This chapter summarizes the pathophysiological roles of Ca2+ and K+ fluxes in normal and cancer cells, by concentrating on several biological systems in which these functions have been studied in depth, such as early embryos, mammalian cell lines, T lymphocytes, gliomas and colorectal cancer cells. A full understanding of the underlying mechanisms will offer a comprehensive view of the ion channel implication in cancer biology and suggest potential pharmacological targets for novel therapeutic approaches in oncology.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Les télomères sont des structures nucléoprotéiques retrouvées aux extrémités des chromosomes eucaryotes linéaires et un certain nombre minimal de répétitions d’ADN télomériques doit être présent afin d’assurer la stabilité du génome. Cependant, la machinerie de réplication conventionnelle est incapable de dupliquer complètement l'ADN télomérique. Chez pratiquement tous les organismes étudiés, ce problème est résolu par une enzyme spécialisée, nommée télomérase, qui est responsable d’ajouter des répétitions télomériques aux extrémités des chromosomes, spécifiquement à la fin de la phase S du cycle cellulaire. La télomérase est une ribonucléoprotéine composée minimalement d’un ARN non codant constitutif, nommé Tlc1 chez Saccharomyces cerevisiae, et d’une sous-unité protéique catalytique avec une activité de transcriptase inverse, Est2 chez la levure, afin de montrer une activité in vitro. Cependant, l’assemblage avec des sous-unités protéiques supplémentaires est essentiel pour la fonction de la télomérase in vivo. Des études des composantes de la télomérase chez S. cerevisiae suggèrent des changements dynamiques dans la composition en protéines au cours de la progression du cycle cellulaire, en accord avec une action spécifique de la télomérase en phase S tardive. Les mécanismes régulant ces changements ne sont pas clairs, de sorte que la composition complète et l'interactome de l'holoenzyme télomérase restent incertains. Le premier objectif de cette thèse était de mettre au point des expériences de criblage à large échelle afin d’étudier la composition et l’interactome de la télomérase selon le cycle cellulaire chez la levure. En plus de déterminer la composition de la télomérase dans chaque phase du cycle cellulaire, nous avons identifié et validé des protéines candidates impliquées dans le transport de l’ARN Tlc1, la biogenèse de la télomérase, et possiblement, dans la régulation des évènements dictant la biologie et la fonction de la télomérase. De plus, des étud, Telomeres are nucleoprotein structures found at the ends of linear eukaryotic chromosomes, and a certain minimum number of telomeric DNA repeats is required to ensure genome stability. However, the conventional replication machinery is unable to completely duplicate telomeric DNA. In virtually all studied organisms, this problem is solved by a specialized enzyme, called telomerase, which is responsible for adding telomeric repeats to the ends of chromosomes, in late S phase of the cell cycle. Telomerase is a ribonucleoprotein (RNP) composed minimally of a constitutive non-coding RNA, called Tlc1 in Saccharomyces cerevisiae, and a catalytic protein subunit with a reverse transcriptase activity, Est2 in yeast, to show in vitro activity. However, the assembly with additional protein subunits is essential for telomerase function in vivo. Studies of telomerase components in S. cerevisiae suggested dynamic changes in protein composition during cell cycle progression, consistent with a specific action of telomerase in late S phase. The mechanisms regulating these changes are unclear and the complete composition and interactome of the telomerase holoenzyme remain uncertain. The first goal of this thesis was to develop large-scale screening experiments to study the cell cycle-dependent composition and interactome of telomerase in yeast. In addition to determining the composition of telomerase in each phase of the cell cycle, we identified and validated candidate proteins involved in the transport of the Tlc1 RNA, in telomerase biogenesis, and possibly, in the regulation of events dictating telomerase biology and function. Furthermore, studies have shown that newly transcribed Tlc1 RNAs undergo a nucleo-cytoplasmic cycle that is suggested to serve as a quality control mechanism for the generation of a functional telomerase RNP. However, telomerase maturation and biogenesis are complex processes that are not fully understood. The second goal of this thesis was to elu

Homologous recombination (HR) entails the formation of several branched recombination intermediates that must be timely disengaged to safeguard chromosome segregation and cell viability. Hence, cells are endowed with DNA helicases and structure-selective endonucleases (SSEs) that sever these physical connections between DNA joint molecules prior to cell division. Surprinsingly, the activation of SSEs is delayed until the final stages of cell cycle. This strategy, conserved from yeast to humans, suggests that this tight control could be crucial to prevent the unscheduled processing of DNA replication and repair intermediates. To understand the biological relevance of this strict regulatory system, we searched for genetic interactions between a constitutively active version of the YEN1 nuclease (YEN1-ON) and key helicases involved in DNA replication and repair. Here, we show that deletion of the conserved PIF1 helicase in a YEN1-ON strain results in a dramatic reduction of its viability under genotoxic stress. This suggests that the unscheduled nucleolytic processing of secondary DNA structures accumulated in the absence of PIF1 is detrimental for cells. PIF1 encodes both mitochondrial and nuclear isoforms of the enzyme, but widely employed separation-of-function alleles (nuclear or mitochondrial-specific) failed to recapitulate such genetic interaction. This prompted us to delve into the translational mechanism of Pif1, leading us to the refinement of the molecular mechanism of alternative translation initiation for PIF1 mRNA, the discovery of a new nuclear Pif1 isoform and the development of the first bona-fide nuclear-null Pif1 isoform.

Cellular responses to DNA damage constitute one of the most important fields in cancer biology. In this class we will analyze classical and recent papers from the primary research literature to gain a profound understand of cell cycle regulation and DNA damage checkpoints that act as powerful emergency brakes to prevent cancer. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. In 1971, President Nixon declared the "War on Cancer," but after three decades the war is still raging. How much progress have we made toward winning the war and what are we doing to improve the fight? Understanding the molecular and cellular events involved in tumor formation, progression, and metastasis is crucial to the development of innovative therapy for cancer patients. Insights into these processes have been gleaned through basic research using biochemical, molecular, and genetic analysis in yeast, C. elegans, mice, and cell culture models. We will explore the laboratory tools and techniques used to perform cancer research, major discoveries in cancer biology, and the medical implications of these breakthroughs. A focus of the class will be critical analysis of the primary literature to foster understanding of the strengths and limitations of various approaches to cancer research. Special attention will be made to the clinical implications of cancer research performed in model organisms and the prospects for ending the battle with this devastating disease.

"Emerging Trends in Agricultural Economics and Extension" is a groundbreaking book that delves into the dynamic and evolving field of agricultural economics and extension. This comprehensive volume explores the latest developments, challenges, and opportunities in agriculture, shedding light on the critical role of economics and extension services in shaping the future of farming and food security. It provides a comprehensive and up-to-date exploration of the multifaceted field of agricultural economics and extension. This book is essential reading for students, researchers, policymakers, and anyone interested in the future of agriculture and the critical role that economics and extension services play in ensuring food security and sustainable farming practices. It offers valuable insights into the evolving challenges and opportunities in agriculture, paving the way for a more resilient and prosperous agricultural sector. Economics, often referred to as the "dismal science," is the study of how societies allocate their scarce resources to meet their diverse needs and desires. It forms the intellectual foundation for understanding how individuals, businesses, governments, and societies make choices in the face of limited resources. Economics is not just about numbers and graphs; it's about the fundamental decisions that shape our world. Extension, on the other hand, is the conduit through which knowledge and innovation flow from experts, researchers, and institutions to the grassroots level. It is the bridge that connects the latest advancements in various fields, including agriculture, community development, health, and education, to those who can benefit most from these innovations. Extension professionals are the unsung heroes who empower individuals and communities to make informed choices, adapt to change, and improve their quality of life. We extend our gratitude to the experts, practitioners, and scholars who have contributed their knowledge and insights to ma

DEAD-box RNA helicases regulate each stage of the RNA life-cycle during gene expression. Ded1 is an essential yeast DEAD-box protein that regulates translation initiation through its effects on mRNA secondary structure and formation of pre-initiation complexes. Ded1 binding to mRNA is not sequence specific, and therefore, it relies on interaction partners for its specificity and regulatory activities during initiation. Stress conditions require large-scale changes in translation that upregulate certain stress response genes but repress most other nonstress-related genes. The target-of-rapamycin (TOR) pathway is a major regulator of these changes, and we have found that Ded1 is a critical mediator of this stress response. Interestingly, in contrast to its role in promoting translation initiation in pro-growth conditions, Ded1 plays an active role in repressing translation upon TOR inactivation. My work focuses on further characterizing the currently unknown interactions critical for Ded1’s repressive function during cellular stress. My results support a physical interaction between Ded1 and Cdc28 in stress conditions that is absent in normal growth conditions, and follow-up results suggest that this interaction may help to coordinate the cell cycle and translation during stress. Along with this clear connection to Cdc28, I conducted a large-scale screen that also shows connections of Ded1 with ATP transport, stress granule formation, cellular localization, cellular trafficking, and mitochondrial translation. All of these could play a key role in understanding how Ded1 fits into the larger picture of translational regulation during TOR inactivation and each subset seen in the annotated GO terms could be an individual area of study for future understanding of stress responses and translation.

Rigosertib is a small molecule in preclinical development that, due to its characteristics as a dual PLK1 and PI3K inhibitor, is particularly effective in counteracting the advance of different types of tumors. In this work, we evaluated the efficacy of Rigosertib and the expression of p53 in five different human tumor cell lines in vitro, A549 (lung adenocarcinoma), MCF-7 and MDA-MB231 (breast cancer cells), RPMI 8226 (multiple myeloma), and U87-MG (glioblastoma). We demonstrated that in all cell lines, the effect was dose- and time-dependent, but A549 cells were the most sensible to the treatment while higher concentrations were required for the most resistant cell line U87-MG. Moreover, the highest and lowest p53 levels have been observed, respectively, in A459 and U87-MG cells. The alterations in the cell cycle and in cell-cycle-related proteins were observed in A549 at lower concentrations than U87-MG. In conclusion, with this article we have demonstrated that Rigosertib has different efficacy depending on the cell line considered and that it could be a potential antineoplastic agent against lung cancer in humans.

The endoplasmic reticulum (ER) orchestrates different cellular processes by which proteins are synthesized, correctly folded, modified and ultimately transported to their final destinations. As part of this crucial biosynthetic process, proteins that are not properly folded and consequently detrimental to normal cellular function are constantly generated. A common signature of many neurodegenerative diseases, including Alzheimer's and Parkinson's, is accumulation and deposition of misfolded proteins that arise when the ability of cells to deal with the burden of misfolded proteins is compromised. In this course, we will explore how the ER quality control machinery ensures that only properly assembled proteins exit the ER while distinguishing between nascent proteins en route to their biologically active folded state from those that are terminally misfolded.

Cells, regardless of whether they are in an organ in the human body or a component of a bacterial colony, can sense the chemical composition of the environment, the presence of neighboring cells, and even the types of their neighboring cells. Depending on the identity of a cell and the information it receives from its environment, it can grow (increase in size), proliferate (make more cells), become quiescent (stop growing and dividing), differentiate (make different types of cells), or die. How cells achieve the astonishing feat of appropriately sensing and responding to their environment has been a major question in biology. In this course we will read and critically discuss the primary scientific literature with the goal of highlighting the basic principles of cell growth, adaptation, and differentiation. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

In this class we will learn about how the process of DNA replication is regulated throughout the cell cycle and what happens when DNA replication goes awry. How does the cell know when and where to begin replicating its DNA? How does a cell prevent its DNA from being replicated more than once? How does damaged DNA cause the cell to arrest DNA replication until that damage has been repaired? And how is the duplication of the genome coordinated with other essential processes? We will examine both classical and current papers from the scientific literature to provide answers to these questions and to gain insights into how biologists have approached such problems. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

In this class we will learn about how the process of DNA replication is regulated throughout the cell cycle and what happens when DNA replication goes awry. How does the cell know when and where to begin replicating its DNA? How does a cell prevent its DNA from being replicated more than once? How does damaged DNA cause the cell to arrest DNA replication until that damage has been repaired? And how is the duplication of the genome coordinated with other essential processes? We will examine both classical and current papers from the scientific literature to provide answers to these questions and to gain insights into how biologists have approached such problems. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.