Biotechnology

Summary

Biotechnology is a multidisciplinary field that involves the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term biotechnology was first used by Károly Ereky in 1919, to refer to the production of products from raw materials with the aid of living organisms. The core principle of biotechnology involves harnessing biological systems and organisms, such as bacteria, yeast, and plants, to perform specific tasks or produce valuable substances. Biotechnology had a significant impact on many areas of society, from medicine to agriculture to environmental science. One of the key techniques used in biotechnology is genetic engineering, which allows scientists to modify the genetic makeup of organisms to achieve desired outcomes. This can involve inserting genes from one organism into another, creating new traits or modifying existing ones. Other importan

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https://en.wikipedia.org/wiki/Biotechnology

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Development of an in vitro microenvironment for maturing oocytes

Jong Eun Ihm

The development of in vitro culture systems, comparable to the in vivo microenvironment in terms of effect on the oocyte growth and development could provide a valuable experimental tool for studying the mechanisms governing folliculogenesis. This tool might serve as well for practical clinical, agricultural, zoological, or biotechnological applications. This thesis reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only served as a scaffold, but it was also used a "reservoir" of immobilized cues. Tethered integrin-binding peptides in combination with other signaling factors aimed at better understanding the interactions of the oocyte and its surrounding granulosa cells that, most probably, determine the efficiency of the actual and the future in vitro mature oocyte production strategies. This work led also to investigating the lymph angiogenesis and the lymphatic transport in the context of oocyte maturation and their impact on mice fertility. In a first step, the mechanical properties of PEG-hydrogel were optimized for culturing secondary follicles. This report shows that the growth of the follicles was highly dependent on the mechanical properties of the surrounding environment. The optimal elastic modulus was found to be close to 900 Pa. In a second step, the effect of Arg-Gly-Asp (RGD) peptide, the minimal integrin-binding sequence, was studied. RGD presence did not influence the follicle growth rate but it significantly improved the quality of the produced oocytes. These findings demonstrated that approaching, biochemically and mechanically, the complexity of the ovarian extracellular matrix could be a winning strategy. The effect of key soluble factors was also investigated in order 1- to confirm their compatibility with the established 3-D culture system and 2- to further improve qualitatively and quantitatively the produced mature oocytes. Various combinations of gonadotropins such as the follicle-stimulating hormone (FSH) or the luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known effects in vivo. Here the aberrant effects of these hormones in the used 2-D systems appeared clearly. c-Kit ligand (KL) is suspected to be one of the most important factors for the activation of primordial follicles and thus for controlling the exit from the resting pool. Previously, studying the effects of the two forms of KL (soluble and membrane-attached) had to cope with the lack of biologically-relevant immobilization strategies. For overcoming this problem, KL constructs were designed to include a substrate sequence for Factor XIIIa (NQEQVSPL or NQEQVSPLRCG). Thus the produced recombinant KL proteins could be enzymatically crosslinked to the PEG matrix. The different constructs of KL, including a wild type extracellular domain, were successfully cloned and produced from mammalian HEK-293 cells. The identity and the activity of the produced proteins were confirmed. Ovarian tissues from four days-old mice were cultured in PEG-hydrogels functionalized with KL. The results showed that the primordial follicles grew and were activated in the PEG-hydrogels where KL was immobilized but not when the soluble form of KL was preset in the medium. This experiment showed that the membrane-attached and the soluble form of KL play a different role in the rodent folliculogenesis. In parallel it was observed that blocking vascular endothelial growth factor receptor-3 (VEGFR-3) signaling had a critical but still unsuspected role in reproduction. This study demonstrates that variation in lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary; blocking lymphangiogenesis, might have an effect on hormone transport and thus on pregnancy. The reported results demonstrates that the blockade of lymphangiogenesis decreases the progesterone and estradioal levels during pregnancy and in fine results in failed fetal development. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system might serve as a platform for developing clinical and biotechnology applications.

EPFL2008

Microfluidic iMITOMI platform to study the architecture of low-affinity transcription factor binding site clusters

Ivan Istomin

Gene regulatory networks (GRNs) determine cellular behaviour, and ultimately the functioning of single- and multicellular organisms. Transcription factors regulate gene expression by binding to DNA or via remodelling chromatin. Recent advances in biotechnological methods have made it possible to precisely characterise the binding of transcription factors to single consensus binding sites. Advanced sequencing methods allow the efficient discovery of de novo binding consensus sequences, and thus provide insight into the evolution of GRNs. However, clusters of transcription factor binding sites have been poorly studied despite their prevalence in eukaryotic genomes. Widely-used methods of GRN interrogation lack the sensitivity for transient binding events with low-affinity binding sites. In my PhD, using the MITOMI platform I showed that two orthologs of the transcription factor Pho4 from S. cerevisiae and C. glabrata that have evolved distinct regulation have similar 3D structure and DNA binding preferences over a wide affinity range. I have developed the iMITOMI assay that preserves the unique advantages of the original MITOMI platform, and thus too has the ability to rapidly characterise transient binding events with high-throughput. In addition, iMITOMI enables the study of transcription factor binding site clusters. We have studied in detail the interactions of Pho4 and Zif268 with clusters of one to six binding sites. The individual binding sites had affinities one to two orders of magnitude below the affinity of the corresponding consensus sequence. We discovered that clusters of low-affinity binding sites could give rise to transcription factor occupancies comparable to or higher than those of single-consensus sequences. Our results demonstrate that low-affinity binding site clusters give rise to high occupancy levels. These have significant implications for the biophysical characterisation of transcription factor binding, the de novo evolution of enhancers and promoters, and the development of synthetic promoters.

EPFL2020

Proteins are fundamental components in biological systems and crucial for a variety of biological functions. Over the last decades, significant progress in structural biology has facilitated the study of proteins and their interactions with molecular partners, shedding light on the relationship between protein structure and function.Progressing beyond the natural protein repertoire, the development of computational methods has enabled the design of novel proteins, primarily focusing on defined structural and biophysical properties. However, consistently introducing new functions into proteins has remained an ambitious challenge. Continuous refinement of computational design methods may prove key to solve emerging biomedical needs. One major challenge where functional protein design could have profound impact is the development of improved vaccines for pathogens that have resisted current vaccine development strategies. Among these pathogens are the influenza and hepatitis C virus (HCV) that cause severe morbidity and mortality across the population. In recent years, several broadly protective antibodies targeting conserved antigenic sites have been isolated and the structural characterization in complex with their cognate epitopes has revealed the structural aspects of these interactions. Computational design of tailor-made immunogens could be an effective tool in inducing a robust neutralizing antibody response against these key antigenic sites.To address these problems, my thesis work introduces a novel surface-centric computational design approach, that focuses on the molecular surface shape and electrostatic properties as means for protein engineering. The surface-centric design approach presents a general framework to design proteins that mimic functional sites, including complex structural motifs characterized by irregular or discontinuous segments. Beyond the design of potent immunogens mimicking epitopes, the protocol allows computational screening for highly specific protein binders and together provide potentially new routes for the design of functional proteins through the optimization of surface features.Building upon the introduced design concepts, my work demonstrates for the first time the successful design of influenza epitope mimetics derived from heterologous protein scaffolds for a conserved, structurally complex antigenic site. The designed immunogens elicited subtype cross-reactive antibodies in vivo and a preliminary study suggests protection of mice from lethal viral infection. Additionally, engineered epitope-specific immunogens for HCV conferred significant cross-neutralization activity against a panel of different HCV genotypes displaying varying neutralization resistance. Diverging from traditional vaccine design approaches, these results provide a general roadmap to engineer novel immunogens eliciting a focused immune response for other pathogens. Overall, the work presented in this thesis introduces a versatile protein design approach for the development of sophisticated functional proteins and showcases the design of precision immunogens. The development of functional proteins could be transformative to solve biomedical questions including not only the design of novel immunogens but also protein components for synthetic biology or other protein-based biotechnologies.

EPFL2021