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Made available in DSpace on 2022-04-28T19:47:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Dixenic parasites often encounter environmental extremes during the transition from vector to host. Preadapted transmission stages overcome these challenges to promote parasites' survival and ensure life cycle progression. Recently, Vigneron et al. and Briggs et al. used single-cell transcriptomics to investigate developmental stage specific gene expression patterns during parasite differentiation. DNA Replication and Repair Laboratory Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) Faculty of Medicine of Ribeirão Preto University of São Paulo The Wellcome Centre for Integrative Parasitology Institute of Infection Immunity and Inflammation University of Glasgow DNA Replication and Repair Laboratory Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) FAPESP: 2019/10753-2 FAPESP: 2020/01883-7 FAPESP: 2020/10277-3 FAPESP: 2020/16480-5

Inheritance of aberrant chromosomes can compromise genome integrity and affect cellular fitness. In eukaryotes, surveillance pathways and cell cycle checkpoints monitor for aberrant DNA transmission and the ATR kinase, a regulator of the DNA damage response, plays a pivotal role. Prior work revealed that ATR acts during antigenic variation in Trypanosoma brucei mammal-infective life cycle forms and that its loss is lethal, but how widely ATR operates in genome maintenance is largely unknown. Here, we show that after prolonged ATR depletion by RNAi T. brucei continues to synthesise DNA and enters new rounds of cell division, despite increased genome damage. Furthermore, we detect defective chromosome segregation, ‘micronuclei’ formation and disruption of the nuclear architecture. RNA-seq revealed that loss of ATR affects the expression of nearly half the genes in the genome, including both RNA Polymerase I and II transcription. Using ChIP-seq of yH2A and DRIP-seq, we reveal overlapping signals for genome damage and R-loops after ATR depletion in all intergenic regions. In addition, we report reduced R-loop levels and accumulation of yH2A signal within centromeres. Together, our data indicates widespread roles of ATR in T. brucei, including differing roles in R-loop homeostasis during multigene transcription and in chromosome segregation.

The genomes of all organisms are read throughout their growth and development, generating new copies during cell division and encoding the cellular activities dictated by the genome’s content. However, genomes are not invariant information stores but are purposefully altered in minor and major ways, adapting cellular behaviour and driving evolution. Kinetoplastids are eukaryotic microbes that display a wide range of such read–write genome activities, in many cases affecting critical aspects of their biology, such as host adaptation. Here we discuss the range of read–write genome changes found in two well-studied kinetoplastid parasites, Trypanosoma brucei and Leishmania, focusing on recent work that suggests such adaptive genome variation is linked to novel strategies the parasites use to replicate their unconventional genomes.

Understanding the rate and patterns of genome variation is becoming ever more amenable to whole-genome analysis through advances in DNA sequencing, which may, at least in some circumstances, have supplanted more localized analyses by cellular and genetic approaches. Whole-genome analyses can utilize both short- and long-read sequence technologies. Here we describe how sequence generated by these approaches has been used in trypanosomatids to examine DNA replication dynamics, the accumulation of modified histone H2A due to genome damage, and evaluation of genome variation, focusing on ploidy change.

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 &plusmn, 0.03 &micro, M), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at &ge, IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.

To evade mammalian immunity, Trypanosoma brucei switches the variant surface glycoprotein (VSG) expressed on its surface. Key to this reaction are controls exerted to ensure only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have to date been implicated only in catalysis of VSG switching by recombination, not transcriptional control. However, how VSG switching is signalled to guide the appropriate reaction, or to integrate switching into parasite growth, is unknown. Here we show that loss of ATR, a DNA damage signalling protein kinase, is lethal and causes increased nuclear genome lesions. ATR depletion also causes expression of mixed VSGs on the cell surface, increased transcription of genes from silent expression sites, and altered localisation of RNA Polymerase I and VEX1, factors involved in VSG transcription. The work therefore reveals that VSG expression control is mediated by a nuclear DNA damage signalling factor.

The magnetic resonance (MR) bore's structure, loading parameters, and material makeup greatly influence the ability of a frequency-specific signal to propagate. The relative path loss behavior of the MR bore has been determined experimentally at ultra-high frequencies (UHFs). An experiment is conducted to determine signal attenuation when the bore is empty (air-filled) or dielectrically loaded (patient-filled). This information is especially important to the wireless network designer who is implementing an in-bore physiological monitoring network. Experimental results show that the MR bore exhibits impressive path loss performance at 434 MHz, with greatest signal strength arriving at the receiver when the bore is dielectrically loaded.