Babesia bovis is a causative agent of bovine babesiosis and is transmitted by vector ticks, Rhipicephalus (Boophilus) spp. The disease has a high mortality rate in susceptible cattle, causing serious economic loss. At present, the only commercial vaccine is culture-based with limited availability. No effective molecular vaccine has been developed to date. Generating a vaccine with specific critical epitopes responsible for protection against B. bovis is critically important. Immunity against B. bovis requires both innate and adaptive responses, with antigen-specific CD4+ T cells essential to the latter through production of IFN-γ. Fourteen B. bovis proteins were selected as putative vaccine candidates and their full-length genes cloned for recombinant protein production intended for evaluating peripheral blood mononuclear cell IFN-γ secretion level from experimentally infected animals in ELISPOT. All proteins expressed in insoluble form (inclusion bodies) and could not be purified. B. bovis genes were then truncated to exclude signal peptide and transmembrane regions, then cloned and expressed using pET101/D-TOPO in Escherichia coli to obtain soluble, useable proteins. Only recombinant B. bovis MSA1, MSA2b and MSA2a1 proteins were successfully expressed in soluble form. These proteins induce invasion-blocking antibodies in immunized cattle, are hypothesized to elicit protection in susceptible animals, but were previously studied by others. Due to failure to produce new candidates to assay, the animal experiments were not performed. Instead, sera from field-infected cattle were assayed for reactivity against the MSA proteins by indirect immunofluorescent antibody (IFA) and western blot (WB) analysis. Field sera from South Texas (#41) and the Mexican Yucatan (#6, #9 and #11) along with positive and negative controls were tested. In IFA test, cattle #6, #9 and #41 were positive while #11 was negative. In WB, #41 and #6 reacted with the recombinant MSA proteins and with control B. bovis whole parasite lysate. However, both #11 and #9 had no signal in WB, although the latter was positive in IFA. Several theories may explain this phenomenon, such as the different preparation process of the antigen in the two tests, strain differences between sera and test antigens, or the different design and nature of each test.

Charles University, Faculty of pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Ženklová Supervisors: Prof. Fabio Altieri and PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Involvement of PDIA3 in oxidative stress response PDIA3 is a member of the protein disulfide isomerase family (PDI) and it is a stress- responsive protein. It is also involved in various cellular signalling pathways and has various functions in the cell. The best-known location is in the endoplasmic reticulum where it plays a major role mainly in the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class I. However, its existence has also been described in many other cell compartments, such as nucleus, mitochondria, cell surface or cytosol, where it interferes in various processes. While in some instances these roles need to be confirmed by further studies, a lot of observations confirmed its involvement in the signal transduction (for example releated with STAT protein) from the cell surface and the regulatory processes in the nucleus. Recent studies have also confirmed its increased expression in various pathological states. The aim of our work was to find out what is its role in the exposure of the MDA-MB 468...

Mitosis is a critically important and time sensitive cellular process that proceeds rapidly, typically completing in 15-45 minutes. Mechanisms have evolved to measure the duration of mitosis, resulting in the identification of aberrant cells that spend too long in mitosis. If non-transformed cells undergo a mitosis that exceeds 90 minutes, then the resulting daughter cells activate a durable G1 arrest and cease proliferating. The underlying mechanism acting to time the duration of mitosis is unknown. Here, we demonstrate that cells activate the Hippo pathway upon entry into mitosis, which initiates degradation of the pro-growth transcriptional co-activators YAP and TAZ. Consequently, prolonged mitosis leads to decreased YAP/TAZ levels in the following G1, thus enforcing cell cycle arrest. We reveal that inactivation of the Hippo pathway, which is common in solid tumors, is sufficient to restore YAP/TAZ levels following a prolonged mitosis, and cells born from this prolonged mitosis can progress through the cell cycle. We also demonstrate that Hippo pathway inactivation alters cell fate decisions in response to mitotic arrest. Antimitotics (e.g. Taxol) have long been used to permanently arrest cells in mitosis, which frequently results in mitotic cell death. It has long been recognized that some cancer cells are resistant to antimitotics; this resistance can arise from cells escaping mitosis into the G1 phase in a process termed mitotic slippage. The mechanisms underlying these cell fate decisions are poorly understood. Here, we demonstrate that inactivation of the Hippo pathway promotes mitotic slippage and overall survival in cells treated with antimitotics by increasing antiapoptotic protein expression. Our data suggest that inactivation of the Hippo pathway may promote resistance to antimitotic therapies by favoring the survival and proliferation of cells that have experienced a prolonged mitosis. Interestingly, we find that restoring Hippo signaling to cancer cells that are resistant to antimitotic therapies sensitizes them to antimitotics and promotes mitotic cell death. Overall, we illuminate a broad role for Hippo signaling in determining cell fate during mitosis and identify a novel mechanism by which resistance to antimitotic therapies can arise. / 2020-10-24T00:00:00Z

The CCR4-NOT complex is a global regulator of gene expression which controls mRNA levels by removing the poly-(A) tail, a step known as deadenylation and one that constitutes the rate-limiting step in mRNA decay. The human complex is comprised of eight stably associated CNOT subunits where CNOT1 forms the scaffold onto which CNOT2-11 bind. Although much has been learnt about the CCR4-NOT complex, questions still remain. Thus, this study focused on a number of sub-complexes of CNOT subunits and associated proteins to determine the mode of interaction with a hope to explore the mechanism of deadenylation by the CCR4-NOT complex. Firstly, the complex of CNOT10:CNOT11, found only in higher eukaryotes, was reconstituted for the first time using recombinant proteins. Crystallisation trials, limited proteolysis and mass spectrometry were used to isolate novel interaction regions between CNOT10 and CNOT11 which may provide direction for future structural and functional studies. Secondly, the interaction between CNOT9 and TTP was characterised. TTP is a RNA-binding protein which targets inflammatory mRNAs for deadenylation by recruiting the CCR4-NOT complex. This study highlights novel interactions between TTP and both CNOT2 and CNOT9. Moreover, BioLayer interferometry (BLI), peptide arrays and site-directed mutagenesis identified that TTP interacts with CNOT9 in a tryptophan-mediated manner. These findings change the known interface between TTP and the CCR4-NOT complex. Lastly, the MultiBac system was used to reconstitute a number of human CNOT sub-complexes, one of which was shown to effectively degrade a poly-(A) substrate, demonstrating it is enzymatically active. This achievement provides a tool for the future study of the CCR4-NOT complex. In summary, this study highlights novel interactions and characterises previously unknown binding mechanisms between CCR4-NOT subunits which expands our current understanding of the complex.

abstract: Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology. / Dissertation/Thesis / M.S. Biochemistry 2012

abstract: The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration. / Dissertation/Thesis / Ph.D. Chemistry 2013

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22665号 / 農博第2420号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5296(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The remarkable metabolic capacity of the soil-dwelling bacterium Sinorhizobium meliloti is encoded on its three circular replicons: the chromosome and two large megaplasmids, pSymA and pSymB. Despite making up 45% of the genome, the pSymA and pSymB megaplasmids can be cured from S. meliloti. This unique attribute provides an opportunity to study the essentiality of chromosomal genes in the presence or absence of nearly half the genome. By interrogating chromosomal genes via massively parallel transposon insertion sequencing (Tn-seq) in the presence and absence of pSymA and pSymB, we identified 307 genes as being essential for viability regardless of the genomic context and 104 genes as being essential specifically when the megaplasmids are absent. We also found that ten percent of genes encoded on the chromosome genetically interact with genes on pSymA and pSymB. In addition, Tn-seq data were utilized to significantly refine a metabolic model of S. meliloti, facilitating more accurate fitness predictions in user-defined nutrient and genetic contexts. Furthermore, the development of a library of barcoded transposon insertion (BarSeq) mutants has enabled us to identify genes that are essential for robust growth in hundreds of nutrient environments simultaneously. This will greatly assist efforts to assign more specific functions to the ~30% of S. meliloti genes that have remained uncharacterized over the years. S. meliloti has been studied for decades as a model organism for symbiotic communication. Its legume host, Medicago truncatula, provides fixed carbon for the bacteria in order to receive fixed nitrogen in return. The molecular dialogue between S. meliloti and M. truncatula, initiates and controls each stage of symbiotic development. When inside host cells, intracellular bacteria are subjected to an arsenal of plant-derived Nodule-specific Cysteine-Rich (NCR) peptides that induce significant morphological changes prior to nitrogen fixation. It was previously shown that a bacterial peptidase, HrrP, present in about 10% of S. meliloti isolates, could degrade host-derived peptides and give the bacterial symbionts greater fitness at the expense of the host. In a screen through peptidases conserved throughout the core S. meliloti genome, we identified one peptidase (sapA) that, when overexpressed, significantly modulates symbiotic outcome. In a manner similar to HrrP, SapA degrades NCR peptides in vitro. Additionally, expression of sapA seems to occur specifically inside the plant host providing compelling evidence that some rhizobial peptidases may have evolved away from housekeeping and toward symbiotic functions.

It is estimated that one in 59 children in the US are affected by autism spectrum disorder (ASD). ASD is distinguished by social and communication deficits that can be displayed throughout a wide range of severity. This resulting spectrum of behaviors observed in ASD suggests that a complex etiology is involved. Previous studies have shown a genetic susceptibility to autism including paternal age, twin and sibling concordance. Genetic sequencing of those affected as well as first order relatives have identified alterations in genes associated with neuronal synaptic communication. However, very little information is available regarding the pathophysiology of synapses in ASD. Neuronal communication between anterior cingulate cortical neurons via synapses with other brain regions is vital in the execution of social behaviors in individuals. The aim of this study was to evaluate the protein expression of the synaptic marker spinophilin and post-synaptic density protein-95 (PSD-95) in postmortem ASD gray matter brain tissue from the anterior cingulate and frontal cortex to compare to typically developing (TD) control brain tissue. Postmortem brain tissue of ASD and TD subjects was acquired from nationally funded brain repositories previously matched by brain area, age and gender. Immunoblotting for spinophilin and PSD-95 was performed using anterior cingulate and frontal cortical gray matter brain tissue from matched ASD and TD brain tissue. Spinophilin and PSD-95 protein amounts for all donors were normalized using GAPDH. Frontal cortical tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=6). Anterior cingulate tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=5). PSD-95 protein expression levels did not result in any significant differences between ASD donors and their control pairs for either brain tissue region. Although no changes were detected in the frontal cortex or anterior cingulate cortex, more brain areas and subjects must be evaluated to determine if spinophilin or PSD-95 can be reliable markers for synaptic alterations in ASD. These data are critical in determining synaptic pathology in ASD which may lead to future treatments.

The GPS2 protein (G-protein pathway suppressor 2) is a product of the mammalian gps2 gene. It was originally identified and characterized in the context of G protein mitogen-activated protein kinase (MAPK) signaling pathways. Several studies have linked GPS2 with the inhibition of the ubiquitin conjugating enzyme UBC13. GPS2-mediated inhibition of UBC13 regulates several metabolic and inflammatory pathways. It has been shown that a lack of GPS2 is correlated with an increase in adiposity and inflammation due to the aberrant activity of UBC13 affected pathways. Therefore, understanding the relationship between UBC13 and GPS2 will provide further understanding of the molecular processes involved in adipose tissue levels, inflammation and downstream molecular responses. In this study, we attempt to determine the molecular determinants of GPS2 interaction with UBC13 by optimizing the protein expression protocol required to produce GPS2 protein expression in Escherichia coli in quantities viable for biochemical and structural assays. Our results indicate that optimization of the gps2 sequence is required for efficient GPS2 protein expression in E. coli cells. Thanks to this optimization we have been able to successfully express GPS2 full length and several fragments, however, further optimization will be required for assessing GPS2-UBC13 molecular binding via in vitro binding assays.