Kinome-wide CRISPR-Cas9 screen to identify kinases involved in Taxol resistance in breast cancer

Breast cancer is the most frequently diagnosed cancer in women, accounting for over 25% of cancer diagnoses and 13% of cancer-related deaths in Canadian women. There are many types of therapies for treatment or management of breast cancer, with chemotherapy being one of the most widely used. Taxol (paclitaxel) is one of the most extensively used chemotherapeutic agents for treating cancers of the breast and numerous other sites. Taxol stabilizes microtubules during mitosis, causing the cell cycle to arrest until eventually the cell undergoes apoptosis. Although Taxol has had significant benefits in many patients, response rates range from only 25-69%, and over half of Taxol-treated patients eventually acquire resistance to the drug. Drug resistance remains one of the greatest barriers to effective cancer treatment, yet little has been discerned regarding resistance to Taxol, despite its widespread clinical use. Kinases are known to be heavily involved in cancer development and progression, and several kinases have been linked to resistance of Taxol and other chemotherapeutic agents. However, a systematic screen for kinases regulating Taxol resistance is lacking. Thus, in this study, a set of kinome-wide screens was conducted to interrogate the involvement of kinases in the Taxol response. Positive-selection and negative-selection CRISPR-Cas9 screens were conducted, whereby a pooled library of 5070 sgRNAs targeted 507 kinase-encoding genes in MCF-7 breast cancer cells that were Taxol-sensitive (WT) or Taxol-resistant (TxR) which were then treated with Taxol. Next generation sequencing (NGS) was performed on cells that survived Taxol treatment, allowing identification and quantitation of sgRNAs. STK38, Blk, FASTK and Nek3 stand out as potentially critical kinases for Taxol-induced apoptosis to occur. Furthermore, kinases CDKL1 and FRK may have a role in Taxol resistance. Further validation of these candidate kinases will provide novel pre-clinical data about potential predictive biomarkers or therapeutic targets for breast cancer patients in the future.

From global advocacy to local resistance. Benin's experience of competency-based school curriculum

In the vein of the "Education for All" campaign to promote access to education, a wave of curriculum revision along the competency-based approach has swept francophone countries in sub-Sahara Africa, thus Benin. The current study documents local actors' various interactions with the curricular reform in the course of its implementation. Secondary data supplemented with qualitative research techniques such as semi-structured interviews with teachers, and focus group discussions with parents enable to relate the patterns of change, the challenges and resistance to change. The actors spectrum generated illustrates advocacy on one hand and resistance on the other. Advocacy of local actors reflects the global optimistic discourse on education and resistance is favoured by disappointing policy outcomes as well as contextual constraints. (DIPF/Orig.)

DATA DRIVEN APPROACHES TO IDENTIFY DETERMINANTS OF HEART DISEASES AND CANCER RESISTANCE

Cancer and cardio-vascular diseases are the leading causes of death world-wide. Caused by systemic genetic and molecular disruptions in cells, these disorders are the manifestation of profound disturbance of normal cellular homeostasis. People suffering or at high risk for these disorders need early diagnosis and personalized therapeutic intervention. Successful implementation of such clinical measures can significantly improve global health. However, development of effective therapies is hindered by the challenges in identifying genetic and molecular determinants of the onset of diseases; and in cases where therapies already exist, the main challenge is to identify molecular determinants that drive resistance to the therapies. Due to the progress in sequencing technologies, the access to a large genome-wide biological data is now extended far beyond few experimental labs to the global research community. The unprecedented availability of the data has revolutionized the capabilities of computational researchers, enabling them to collaboratively address the long standing problems from many different perspectives. Likewise, this thesis tackles the two main public health related challenges using data driven approaches. Numerous association studies have been proposed to identify genomic variants that determine disease. However, their clinical utility remains limited due to their inability to distinguish causal variants from associated variants. In the presented thesis, we first propose a simple scheme that improves association studies in supervised fashion and has shown its applicability in identifying genomic regulatory variants associated with hypertension. Next, we propose a coupled Bayesian regression approach -- eQTeL, which leverages epigenetic data to estimate regulatory and gene interaction potential, and identifies combinations of regulatory genomic variants that explain the gene expression variance. On human heart data, eQTeL not only explains a significantly greater proportion of expression variance in samples, but also predicts gene expression more accurately than other methods. We demonstrate that eQTeL accurately detects causal regulatory SNPs by simulation, particularly those with small effect sizes. Using various functional data, we show that SNPs detected by eQTeL are enriched for allele-specific protein binding and histone modifications, which potentially disrupt binding of core cardiac transcription factors and are spatially proximal to their target. eQTeL SNPs capture a substantial proportion of genetic determinants of expression variance and we estimate that 58% of these SNPs are putatively causal. The challenge of identifying molecular determinants of cancer resistance so far could only be dealt with labor intensive and costly experimental studies, and in case of experimental drugs such studies are infeasible. Here we take a fundamentally different data driven approach to understand the evolving landscape of emerging resistance. We introduce a novel class of genetic interactions termed synthetic rescues (SR) in cancer, which denotes a functional interaction between two genes where a change in the activity of one vulnerable gene (which may be a target of a cancer drug) is lethal, but subsequently altered activity of its partner rescuer gene restores cell viability. Next we describe a comprehensive computational framework --termed INCISOR-- for identifying SR underlying cancer resistance. Applying INCISOR to mine The Cancer Genome Atlas (TCGA), a large collection of cancer patient data, we identified the first pan-cancer SR networks, composed of interactions common to many cancer types. We experimentally test and validate a subset of these interactions involving the master regulator gene mTOR. We find that rescuer genes become increasingly activated as breast cancer progresses, testifying to pervasive ongoing rescue processes. We show that SRs can be utilized to successfully predict patients' survival and response to the majority of current cancer drugs, and importantly, for predicting the emergence of drug resistance from the initial tumor biopsy. Our analysis suggests a potential new strategy for enhancing the effectiveness of existing cancer therapies by targeting their rescuer genes to counteract resistance. The thesis provides statistical frameworks that can harness ever increasing high throughput genomic data to address challenges in determining the molecular underpinnings of hypertension, cardiovascular disease and cancer resistance. We discover novel molecular mechanistic insights that will advance the progress in early disease prevention and personalized therapeutics. Our analyses sheds light on the fundamental biological understanding of gene regulation and interaction, and opens up exciting avenues of translational applications in risk prediction and therapeutics.

Iron metabolism: a double-edged sword in the resistance of glioblastoma to therapies

International audience

Toward an effective strategy in glioblastoma treatment. Part I: resistance mechanisms and strategies to overcome resistance of glioblastoma to temozolomide.

International audience

Identification of resistance factors to the main wheat diseases in the Mediterranean area

Several diseases challenge bread and durum wheat productions worldwide. The importance of these cereals requires adequate protection to pathogens that can cause strong yield and grain quality losses. The main work of this thesis was related to phenotype GDP (Global Durum Panel) in the Mediterranean region (Italy, Egypt, Lebanon, Morocco and Turkey) and Argentina across three years (2019-2021) for yellow rust resistance (infection type and severity). GWAS shows in particular, loci in chromosome 1B, 2B, 4B, 5A, 6A, 7B showed high significance across nurseries/years, with various patterns of GxE. The second chapter is about Zymoseptoria tritici, agent of STB (Septoria Tritici Blotch), a foliar pathogen that yearly causes high damages if not controlled. In recent years research in durum wheat breeding is focused on the identification of novel, underexploited resistance genes to be subsequently and conveniently moved into the pre-breeding and breeding stream. The plants were phenotyped for disease height characters, infection type at the flag leaf and infection type at the level of the canopy below the flag leaf. This experiment opens up a rich scenario of analysis and opportunities to investigate and discover new loci of resistance to STB. Third chapter is about Fusarium head blight (FHB) is a fungal disease caused by pathogens belonging to the genus Fusarium. In particular, Fusarium culmorum and Fusarium graminearum species cause severe grain yield losses and accumulation of mycotoxins in wheat that compromise food safety. Over 250 QTL/genes for FHB resistance have been identified in bread wheat, such as Fhb 1 and Fhb 5 but only a small number of FHB resistance loci have been mapped in durum wheat. The aim of this work is to find loci of partial resistance to FHB already present in durum and bread wheat germplasm and therefore easily cumulative.

Resistance and opposition to the Macedonian kingship in the ancient Iranian world

This thesis deals with the criticism of Macedonian kingship in the ancient Iranian world. The question of indigenous opposition and resistance to the Greeks and Macedonians has been little addressed by ancient historians. The study therefore adopts a different, interdisciplinary perspective and seeks to understand where the utterly negative portrayal of Alexander and the Macedonians found in most Iranian sources stems from. The first part deals with the subject by first examining the acts of violence committed by Alexander and his men against the Iranians during the expedition to Asia that might have led to such a portrayal in the Iranian sources. I have focused on looting, massacres and insults to deities, such as the looting of temples or the destruction of many settlements in ancient Iran handed down in classical sources. To this end, an important part was devoted to the analysis of archaeological sources, especially the signs of destruction in areas such as Persia and Sogdiana. In the second part, the image of Alexander and his successors, although mentioned much less frequently, as it appears in pre-Islamic Iranian literature, is analysed in detail, focusing on the faults and cruelties attributed to them against the Iranians, but especially against their religion. These are mostly Zoroastrian religious sources, whose clergy preserved a demonic image of the Macedonian kings. In the third and final part, further examples of offences committed by the Diadochi and Seleucids against the Iranians in the classical tradition are collected. At the same time, it is examined how the Hellenistic rulers of Iranian origin, e.g. the Arsacids and the Orontids, opposed not only militarily but also ideologically the Macedonian tradition represented by the kingdoms of Macedonian descent and chose a pro-Iranian tradition that was clearly different from the Greco-Roman one.

Motivating Medical Students To Learn Basic Science Concepts Using Chronic Myeloid Leukemia As An Integration Theme.

To report on the use of chronic myeloid leukemia as a theme of basic clinical integration for first year medical students to motivate and enable in-depth understanding of the basic sciences of the future physician. During the past thirteen years we have reviewed and updated the curriculum of the medical school of the Universidade Estadual de Campinas. The main objective of the new curriculum is to teach the students how to learn to learn. Since then, a case of chronic myeloid leukemia has been introduced to first year medical students and discussed in horizontal integration with all themes taught during a molecular and cell biology course. Cell structure and components, protein, chromosomes, gene organization, proliferation, cell cycle, apoptosis, signaling and so on are all themes approached during this course. At the end of every topic approached, the students prepare in advance the corresponding topic of clinical cases chosen randomly during the class, which are then presented by them. During the final class, a paper regarding mutations in the abl gene that cause resistance to tyrosine kinase inhibitors is discussed. After each class, three tests are solved in an interactive evaluation. The course has been successful since its beginning, 13 years ago. Great motivation of those who participated in the course was observed. There were less than 20% absences in the classes. At least three (and as many as nine) students every year were interested in starting research training in the field of hematology. At the end of each class, an interactive evaluation was performed and more than 70% of the answers were correct in each evaluation. Moreover, for the final evaluation, the students summarized, in a written report, the molecular and therapeutic basis of chronic myeloid leukemia, with scores ranging from 0 to 10. Considering all 13 years, a median of 78% of the class scored above 5 (min 74%-max 85%), and a median of 67% scored above 7. Chronic myeloid leukemia is an excellent example of a disease that can be used for clinical basic integration as this disorder involves well known protein, cytogenetic and cell function abnormalities, has well-defined diagnostic strategies and a target oriented therapy.

Impact and fracture resistance of an experimental acrylic polymer with elastomer in different proportions

The purpose of this study was to evaluate the impact and fracture resistance of acrylic resins: a heat-polymerized resin, a high-impact resin and an experimental polymethyl methacrylate with elastomer in different proportions (10, 20, 40 and 60%). 120 specimens were fabricated and submitted to conventional heat-polymerization. For impact test, a Charpy-type impact tester was used. Fracture resistance was assessed with a 3-point bending test by using a mechanical testing machine. Ten specimens were used for each test. Fracture (MPa) and impact resistance values (J.m-1) were submitted to ANOVA - Bonferroni's test - 5% significance level. Materials with higher amount of elastomer had statistically significant differences regarding to impact resistance (p < 0.05). Fracture resistance was superior (p < 0.01) for high-resistance acrylic resin. The increase in elastomer concentration added to polymethyl methacrylate raised the impact resistance and decreased the fracture resistance. Processing the material by injection decreased its resistance to impact and fracture.

Determination of banana fruit susceptibility to post-harvest diseases: wound anthracnose, quiescent anthracnose and crown rot

Introduction. This protocol aims at ( a) evaluating the resistance to post-harvest diseases within different genotypes of bananas, and ( b) comparing different origins of bananas ( geographic origin, physiological stage, etc.) for their susceptibility to post-harvest diseases. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. Materials required and details of the twelve steps of the protocol ( fruit sampling and inoculum preparation, wound anthracnose resistance study, quiescent anthracnose resistance study and crown-rot resistance study) are described. Results. Typical symptoms of the different diseases are obtained after artificial inoculation.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

Background: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

Novel sources of witches` broom resistance (causal agent Moniliophthora perniciosa) from natural populations of Theobroma cacao from the Brazilian Amazon

Witches` broom is a severe disease of Theobroma cacao L. (cacao), caused by the basidiomycete Moniliophthora perniciosa. The use of resistant cultivars is the ultimate method of control, but there are limited sources of resistance. Further, resistance from the most widely used source (`Scavina 6`) has been overcome after a few years of deployment. New sources of resistance have been intensively searched for in the Amazon basin. Here, we evaluated for witches` broom resistance, cacao accessions from various natural cacao populations originally collected in the Brazilian Amazon. Resistance of 43 families was evaluated under nursery and/or field conditions by artificial or natural infection, respectively, based on disease incidence. Screening for resistance by artificial inoculation under nursery conditions appeared to be efficient in identifying these novel resistance sources, confirmed by natural field evaluation over a nine-year period. The increase in natural field infection of `Scavina 6` was clearly demonstrated. Among the evaluated families with the least witches` broom incidence, there were accessions originally collected from distinct river basins, including the Jamari river (`CAB 0371`; `CAB 0388`; `CAB 0392`; and `CAB 0410`); Acre (`CAB 0169`); Javari (`CAB 0352`); Solimes (`CAB 0270`); and from the Purus river basin, the two most outstanding resistant accessions, `CAB 0208` and `CAB 0214`. The large genetic diversity found in cacao populations occurring at river basins from Acre and Amazonas states, Brazil, increased the chance that the selected resistant accessions would be genetically more dissimilar, and represent distinct sources of resistance to M. perniciosa from `Scavina 6`.

QTL mapping for reaction to Phaeosphaeria leaf spot in a tropical maize population

Phaeosphaeria leaf spot (PLS) is an important disease in tropical and subtropical maize (Zea mays, L.) growing areas, but there is limited information on its inheritance. Thus, this research was conducted to study the inheritance of the PLS disease in tropical maize by using QTL mapping and to assess the feasibility of using marker-assisted selection aimed to develop genotypes resistance to this disease. Highly susceptible L14-04B and highly resistant L08-05F inbred lines were crossed to develop an F(2) population. Two-hundred and fifty six F(2) plants were genotyped with 143 microsatellite markers and their F(2:3) progenies were evaluated at seven environments. Ten plants per plot were evaluated 30 days after silk emergence following a rating scale, and the plot means were used for analyses. The heritability coefficient on a progeny mean basis was high (91.37%), and six QTL were mapped, with one QTL on chromosomes 1, 3, 4, and 6, and two QTL on chromosome 8. The gene action of the QTL ranged from additive to partial dominance, and the average level of dominance was partial dominance; also a dominance x dominance epistatic effect was detected between the QTL mapped on chromosome 8. The phenotypic variance explained by each QTL ranged from 2.91 to 11.86%, and the joint QTL effects explained 41.62% of the phenotypic variance. The alleles conditioning resistance to PLS disease of all mapped QTL were in the resistant parental inbred L08-05F. Thus, these alleles could be transferred to other elite maize inbreds by marker-assisted backcross selection to develop hybrids resistant to PLS disease.

Detection of Sourgrass (Digitaria insularis) Biotypes Resistant to Glyphosate in Brazil

Sourgrass is a perennial weed infesting annual and perennial crops in Brazil. Three biotypes (R1, R2, and R3) of sourgrass suspected to be glyphosate-resistant (R) and another one (S) from a natural area without glyphosate application, in Brazil, were tested for resistance to glyphosate based on screening, dose-response, and shikimic acid assays. Both screening and dose-response assays confirmed glyphosate resistance in the three sourgrass biotypes. Dose-response assay indicated a resistance factor of 2.3 for biotype RI and 3.9 for biotypes R2 and R3. The hypothesis of a glyphosate resistance was corroborated on the basis of shikimic acid accumulation, where the S biotype accumulated 3.3, 5.0, and 5.7 times more shikimic acid than biotypes R1, R2, and R3, respectively, 168 h after treatment with 157.50 g ae ha(-1) of glyphosate. There were no differences in contact angle of spray droplets on leaves and spray retention, indicating that differential capture of herbicide by leaves was not responsible for resistance in these biotypes. The results confirmed resistance of sourgrass to glyphosate in Brazil.