Biologie moléculaire | EPFL Graph Search

redresse=1.67|vignette| Géométrie de la double hélice d'ADN B montrant le petit et le grand sillon ainsi que le détail des deux types de paires de bases : thymine–adénine en haut et cytosine–guanine en bas. La biologie moléculaire (parfois abrégée bio. mol.) est une discipline scientifique de la vie au croisement de la génétique, de la biochimie métabolique et de la physique, dont l'objet est la compréhension des mécanismes de fonctionnement de la cellule au niveau moléculaire. Le terme « biologie moléculaire », utilisé la première fois en 1938 par Warren Weaver, désigne également l'ensemble des techniques de manipulation d'acides nucléiques (ADN, ARN), appelées aussi techniques de génie génétique. La biologie moléculaire est apparue au , à la suite de l'élaboration des lois de la génomique, transcriptomique, protéomique, métabolomique) de la biologie moléculaire pour étudier plus spécifiquement les cellules des micro-organismes. Histoire Histoire de la génétique et de la

A new non-toxic chemically cross-linked ELP hydrogel for in situ protein delivery

Karine Ciapala

Proteins play an essential role in all biological processes and have been attracting more and more attention as drug candidates due to their high specificity and activity. However, their use as therapeutics has been seriously held back given that their ability to be effectively and conveniently delivered is problematic. Indeed, simple oral administration is ineffective since proteins are degraded in the digestive tract. Until now, these therapeutic agents were delivered by infusions or frequent injections. But this causes serious problems, as the compound does not get delivered in a prolonged continuous manner. Additionally, injections are also problematic as patients are reluctant to use needles unless there illness is life threatening. That is why focus has grown over the last years on developing novel delivery systems for proteins. Amongst the various types of delivery systems that exist, hydrogel based ones are very promising, as they possess remarkable biomimetic properties. The hydrogels that are chemically cross-linked are stronger than their counterparts, the physical hydrogels. An appealing approach to protein delivery that is more and more sought is to form the hydrogel in situ from an injected aqueous polymer solution and to use the formed gel as a depot for the sustained release of the protein. Oxygen sensitive gellation is an interesting stimulus to form these gels, but has not been successfully developed, as the gelling kinetics are too slow. Therefore, research groups have turned to the use of toxic chemicals to speed up the reaction. Elastin-like poylpeptides (ELP) are artificial biopolymers that are composed of a pentapeptide repeat. This repeat consists of Val-Pro-Gly-Xaa-Gly, where Xaa denotes a guest residue. An interesting characteristic of ELPs is that they are thermally responsive. Meaning, that when in solution they go through a lower critical solution temperature (LCST) transition. Below their LCST they are soluble in water and above their LCST they are insoluble. ELPs are well suited for drug delivery applications for many reasons such as their biocompatibility. The ELP that is studied is ELP9 Gel. Its guest residues are Val, Ala and Cys in a ratio of 1:14:1 respectively. As this ELP has cysteines it has the capacity to form a gel via cross-linking of the thiols groups by oxidation. The rheological tests that were carried out showed that this ELP9 Gel has all the characteristics of a gel. Furthermore, its gelling kinetics were greatly increased by heating it, but still remain too long for in situ applications. This suggests that the LCST behaviour of ELPs seems to play some role in favouring the cross-linking. But further controls remain to be done to confirm this hypothesized tandem process. A delivery strategy that involved this ELP9 Gel and Blue Fluorescent Proteins (BFP) with added cysteines was not successful as the molecular biology to obtain the modified proteins failed. However, the elaboration of a fusion peptide between Glucagon-Like-Peptide-1 (GLP-1) and ELP9 Gel was accomplished. The preliminary activity assay showed a reduced activity in comparison to the GLP-1 peptide alone. Unfortunately this ELP9 Gel has a setback due to its slow gelling time. Therefore, before continuing the research on the delivery strategies, it would be wiser to create a better ELP. If a new ELP with a higher cysteine concentration and lower transition temperature can be created, its gelling kinetics may be favourable for in situ protein delivery applications and thus, a chemically cross-linked hydrogel that does not require any toxic cross-linker would be available. This would be a big step in creating a very robust hydrogel for protein delivery applications but also for tissue regeneration

2009

Integrative transcriptome analyses of Transposable Elements (TEs) and genes contribute to a better understanding of colorectal cancer

Eunji Shin

Cancer is the second leading cause of death worldwide. Cancer develops through multiple hallmark functions including apoptosis evasion, unlimited replicative potential, metastasis, and immune avoidance. Over the past few decades, researchers have reported a substantial amount of information about the role of gene expression dysregulation in cancer, whereas transposable elements (TEs) have been overlooked despite the fact they constitute nearly half of the human genome. TEs are DNA repetitive sequences that spread across the genome of living organisms throughout the evolution of species, contributing to genomic diversity and shaping the epigenomic landscape. The vast majority of TEs appear to be transcriptionally silent in adult tissues, thus avoiding deleterious mutational insertions and recombination events in somatic tissues. Due to the global epigenetic dysregulation that occurs during tumorigenesis, some TEs lose repressive marks and become transcriptionally active in cancer cells. Consequently, TE insertions into oncogenes and tumor suppressor genes may occur, thereby contributing to cancer development and disease progression. It is also widely accepted that some TEs harbor transcription factor recognition sites and have regulatory functions on gene expression. Some transcriptionally active TEs can give rise to alternative TE-derived chimeric gene products. Given these facts, integrative transcriptome analysis of TEs and genes may contribute to a better understanding of complex traits of cancer and help to better classify cancer subtypes with clinical implications for patients.To gain insights into the underlying mechanisms of cancer pathogenesis, we constructed a co-expression network based on the similarity in expression levels of TEs and genes. We focused on colorectal cancer (CRC), a heterogeneous disease with different genetic and molecular backgrounds, contributing to patient outcomes and response to therapy. We investigated the coordinated activity of TEs and genes in view of recurrent CRC chromosomal aberrations, genome organization, and functional significance. We further examined co-expression changes in network structure under the contrasting conditions of cancerous versus matched healthy colic mucosal tissues. We found that the cancer network was associated with a dramatic decrease in the number of gene interactions as opposed to an increase in TE interactions. Physical chromosomal distance affects the degree of co-expression, where the proximal distance effect of TE is contrasted with the distant effect of the gene. Our study sheds light on the complex interplay between TEs and genes and how they coordinately shape cancer and normal co-expression networks. Furthermore, we integrated gene and TE expression to develop a new consensus molecular subtype (CMS) classifier derived from multiple layers of TE and gene signatures. We demonstrated that our new approach led to better identification of CRC patients of the former CMS3 subtype, the most heterogeneous subtype with mixed biological characteristics, and a global reduction of the expected misclassification of other CMS subtypes. We further optimized our classifier for potential clinical use and obtained a set of 50 genes and 20 TE integrants with the best ability to identify CMS subtypes. Our integrative model could be a major add on for the development of future clinical trials by improving patient stratification over current gene-restricted molecular classifications.

EPFL2022

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Elena Aritonovska

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase. Impaired telomerase function can lead to premature organismal aging, development of cancer and multisystem disorders such as dyskeratosis congenita. Telomerase access to telomeres may be regulated by a telomere-binding complex termed shelterin, which is composed of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 proteins. However the molecular basis for human telomerase recruitment to telomeres is not known. Here, we have directly investigated the process of telomerase recruitment via chromatin immunoprecipitation (ChIP) and fluorescence in situ hybridization (FISH). We find that depletion of two components of the shelterin complex – TPP1 and the protein that tethers TPP1 to the complex, TIN2 – results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Furthermore, we find that the double-stranded telomere binding protein, TRF2 is dispensable for telomerase association with telomeres. Deletion of the oligonucleotide/oligosaccharide-binding fold (OB-fold) of TPP1 further disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that the recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere. We propose that the loss of TRF2 can be compensated by the second double-stranded telomere binding protein, TRF1 that anchors TIN2/TPP1-recruited telomerase onto the telomeres. Dyskeratosis congenita (DC) is a multisystem disorder characterized with bone marrow failure, cancer predisposition and defective telomere maintenance. The TINF2 gene that encodes for the TIN2 shelterin protein is one of seven mutated genes identified in DC patients. Here, we have examined the effects of TIN2 DC mutants on telomere protection and shelterin protein stability. We find that exogenous expression of TIN2 DC mutants can rescue the TIN2-depleted phenotype, restoring TPP1 protein levels and telomere protection. Our findings indicate that TIN2 DC mutants preserve the intact telomere structure and protection. We propose that defective telomere elongation may be the underlying cause of impaired telomere maintenance in TIN2 DC patients.

EPFL2011