Biological knowledge management and gene network analysis: a heuristic road to System Biology

In order to understand the molecular basis of living cells and organisms, biologists over the past decades have been studying life's core molecular players: the genes. Most genes have a specific function, a role they play in the collective task of developing a cell and supporting all the aspects of keeping it alive. These genes do not perform their function randomly. Instead, after billions of years of evolution, nature's trial-and-error process, they have become parts of an utterly complex and intricate network, an interconnected mesh of genes that comprises signal detection cascades, enzymatic reactions, control mechanisms, etc. Over several past decades, experimental molecular biologists have sought mainly to study these genes via a one-by-one approach. However, with the advent of high-throughput experimental techniques, the number-crunching power of computers, and the realisation that many biological functions are the result of interactions between genes or their proteins, Biology's related field of Systems Biology has emerged. Here, one tries to combine the dispersed information produced by many researchers, in integrated assemblies called gene networks. Our research comprises the development of two new methods for improved information integration in the field of molecular Systems Biology. The first one aims to support an approach to acquire insights in the dynamics of gene networks (the behaviour of gene activities over time), called 'modelling and simulation' of genetic regulatory networks. Our second new method approaches the problem of how to collect and manage the information necessary to compose such genetic networks in the first place, based on scattered information in a dispersed and increasingly fast growing body of publications. These two methods form two separate parts in this thesis (chapters 2-4, and chapters 5-7). Chapter 1, section 1.3 provides an introductory, complete overview of this thesis. It is intended as a light introduction to my doctoral research, presented in an informal and entertaining way, and mainly addressed to my friends and family. It forms an introduction for the laymen to our work and the concepts that are important for this thesis. Chapters 2, 3 and 4 constitute Part 1 of this thesis. Chapter 2 gives a review of the various formalisms for modelling and simulation of gene networks, as a thorough background for our work presented in the following chapter. Chapter 3 describes SIM-plex, our new software tool that forms a bridge between a mathematical gene network modelling formalism, and the biologist, who usually is more an expert in the biology behind the gene network than a mathematician can ever be. It shields off the mathematics in a new way so as to enable biologists to experiment with modelling and simulation themselves. Chapter 4 describes the various applications that SIM-plex was used for. The research described in Part 2 of this thesis, chapters 5, 6 and 7, emerged from our own need for a better management of biological information. We experienced this necessity while we were building a larger genetic network for the Arabidopsis cell cycle, and it forms a general problem in biology. Chapter 5 gives a background of the currently existing methods for harvesting literature information, but comes to the conclusion that no existing automated or manual method displays sufficient potential to capture the largest part of information from literature in a structured way. In chapter 6, we describe our bold proposal of a new method to tackle this problem: MineMap, a community-based manual text-curation initiative. We describe the various aspects required to make such a project possible, based on our own experiences with our prototype application MineMap. This research is organised in a 'heuristic' way, in the sense that we built a first sketch and a working solution that also generated experiences for improvements in a next design. While chapter 6 describes our new ideas and concrete implementations in considerable detail, chapter 7 then illustrates the core concept behind MineMap

Texture profile analysis reveals a stiffer ovarian cortex after testosterone therapy : a pilot study

Purpose: The importance of the surrounding ovarian stromal cells and extracellular matrix in the development and maturation of follicles has recently gained attention. An aberrant extracellular matrix has been described in ovaries of patients with polycystic ovary syndrome where a more rigid structural environment, possibly induced by endogenous testosterone, impairs normal folliculogenesis. In this context, we describe the textural parameters of the ovarian cortex of transgender men after prolonged testosterone administration compared to the textural parameters of the non-exposed ovarian cortex originating from female oncological patients. Methods: Texture profile analysis (TPA) was performed on ovarian cortex (5 x 5 mm) of oncological and transgender patients in order to measure stiffness, hardness, cohesiveness, and springiness of the ovarian cortex (LRXplus universal testing system). Statistical analysis was performed using repeated measurements mixed models and the Spearman rank order correlation test (IBM SPSS Statistics 23). Results: A total of 36 frozen-thawed cortical strips (5 x 5 mm) were subjected to TPA. The superficial part of cortex fragments originating from transgender persons (fragments < 1.4 mm; N = 10) appeared to be significantly stiffer compared to cortex derived from oncology patients (fragments < 1.4 mm; N = 7) (6.78 +/- 1.38 N/mm versus 5.41 +/- 0.9 N/mm respectively, p = 0.036). Conclusions: This is the first application of TPA in ovarian cortex to study the physical properties. Comparing the physical properties, we objectively describe an increased cortical stiffness in the most outer part of the ovarian cortex following prolonged testosterone administration in transgender men compared to the ovarian cortex of oncological patients. This preliminary and novel approach could be the start of future research to understand the physical properties of ovarian tissue

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ

World Association for the Advancement of Veterinary Parasitology (WAAVP) guideline: anthelmintic combination products targeting nematode infections of ruminants and horses

Increasing threats from anthelmintic resistant nematode populations warrant and motivate a reappraisal of chemotherapeutic strategies for nematode control in ruminant livestock and horses. The objective of this paper is to present a guideline for the evaluation of products containing two or more constituent anthelmintic actives in a single dosage form for the treatment of nematode infections in these animals. At present, regulatory policies on the approval of such products vary across jurisdictions, and this World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline should enable the harmonization of the requirements. This guideline makes clear recommendations on the minimal standards needed, but stresses that registration dossiers for combination anthelmintic products submitted for approval must conform to the standards and practices already established in existing guidelines for anthelmintics

First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides : a pilot study

Background Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs;Ascaris lumbricoides,Trichuris trichiura,Necator americanus,Ancylostoma duodenaleandA.ceylanicum),Strongyloides stercoralisandSchistosomain human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. Methods and principal findings A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris,Trichuris,N.americanus,Ancylostoma,StrongyloidesandSchistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. Conclusions/Significance We showed the technical feasibility of an international EQAS for the NAAT of STHs,StrongyloidesandSchistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs. Author summary Tests that detect parasite DNA in human stool are increasingly being used for the diagnosis of infections with intestinal worms, including schistosomiasis. To ensure the quality in diagnostic testing of these parasitic worms, it is important that laboratories evaluate the diagnostic performance of their DNA-based tests. This can best be achieved by participating in an external quality assessment scheme (EQAS). An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. Currently, such an EQAS for parasitic worms is lacking. We therefore piloted an international EQAS for the diagnosis of parasitic worms involving 15 laboratories in Africa, Asia, Australia, Europe, and North America. Although most laboratories performed well, we could clearly identify those laboratories that may need to improve their test protocol. We found that laboratories were using many different test protocols, and further research should aim to verify whether this has an impact on the performance of the diagnostic outcomes

Phenolic Polymers as Model Melanins

Melanins are a class of conjugated biopolymers with varying compositions and functions, which have a variety of potential medical and technical applications. Here we examine the conjugated polymers derived from a variety of phenolic monomers (catechol (CAT), levodopa (DOPA) and homogentisic acid (HGA)), using a selection of different analytical chemistry techniques to compare their properties with a view to understanding structure-function relations. The polymers displayed measurable conductivity, with electronic properties tuned by the functional groups pendant on the polymer backbones (which served as dopants) suggesting their potential for application in electronic devices