Blood Metabolic and Hematology Parameters and Survivorship in Mice after Application of the Rabies Challenge Virus Standard in Vaccine Potency Test

Background: Rabies virus can cause intensive and lethal infection of the central nervous system (CNS) in animals and humans. Metabolic examinations are conducted at the cerebrospinal fluid (CSF), and it has been found that many metabolic changes occur during RABV infection. However, although it is a neurotropic virus, it can cause damage to extraneural tissues - lungs, heart, kidneys and liver. This study aimed to determine differences in metabolic, endocrinology and hematologic parameters in blood of mice after application of rabies challenge with virus standard 27 strain (CVS-27).Materials, Methods & Results: This study included 30 survived, and 30 dead mice that were part of the standard procedure of NIH (National Institute of Health) test in Pasteur Institute in Novi Sad. Tests were performed in the following order: two groups of mice were vaccinated in a 7 day period with different dilutions of standard vaccine and the examined vaccine. Seven days after the last vaccination, immunized animals and animals in the control group received test virus CVS-27. Blood samples were collected from a heart puncture. Differences in hematologic and biochemical parameters were determined by t-test. Due to a high number of blood parameters, we performed a joint analysis of multiple dependent variables. Higher pH value and higher concentrations of glucose, cholesterol, lactate dehydrogenase (LDH), creatine kinase (CK), albumin, urea, creatinine, α-amylase, magnesium (Mg), nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHB) and lactate were noted in dead mice. Higher granulocytes and mean platelet volume (MPV) were noted in mice which died, but also reduced lymphocytes, erythrocytes, haemoglobin, hematocrit and platelets count. Higher values of insulin, cortisol and HOMA-IR (homeostatic model assessment insulin resistance) were noted in the group of dead mice compared to the surviving one. Reduced QUICKI (quantitative insulin sensitivity check index) value was noted in mice which died compared to the surviving group. Principal component analysis (PCA) showed that components 1 and 2 explain 38.7 of variance and that these two compounds are enough for the distinction between the animals which dies and those that survived. It was found that the cortisol, insulin, HOMAIR, NEFA, aspartate aminotransferase (AST), lactic acid, LDH and granulocyte could explain the variance of the first component, which highly correlated with the first principal component. Also, pH level, glucose, creatinine, albumin and BHB showed significant importance. A positive correlation was shown between those parameters.Discussion: Mice that died during NIH test after applying CVS-27 expressed more significant stress (higher cortisol level). Disturbances of energy metabolism were noted (more significant catabolism of lipids and insulin resistance), changes of protein metabolism caused by muscle load (urea, creatinine, AST and LDH) and general disturbances of acid-base status (higher pH) and dehydration (increased albumin) were also noted in mice that died. Values of hematologic parameters showed minor influence at total variability and are a bit correlated with metabolic changes. In factor analysis, component 1 was determined from numerous parameters. Correlations between component 1 and cortisol, HOMA-IR, lactates, insulin, AST and LDH were noted. It completely determines survived and dead animals after CVS-27 during NIH test. Disturbances in blood parameters showed an analogy with previous studies of CNS. Given parameters can be very useful in clinical-pathological analysis in RABV infection

The discovery and characterization of NHJ-1, a novel regulator of canonical non-homologous end joining in the nematode «Caenorhabditis elegans»

The physical and informational integrity of DNA is of paramount importance to the survival, development, and reproduction of living organisms. Of the various forms of damage which can arise from the numerous intrinsic and extrinsic forms of genotoxic stress, the most serious are double strand breaks (DSBs). These lesions are dangerous not only because of their mutagenic potential, but because they disrupt the physical continuity of DNA molecules and risk the loss of all genes distal to the break. Repair of DSBs is handled by several distinct pathways, of which the classical non-homologous end joining (cNHEJ) is the most prominent in somatic tissues. The mechanism of cNHEJ involves three steps: 1) the rapid recognition and binding of the free DNA end by the heterodimeric Ku ring protein; 2) the processing of the damaged DNA ends by a complement of nucleases, kinases, phosphatases, and polymerases to yield ligation-compatible ends; and 3) the terminal ligation by Ligase IV. Despite the wide conservation of cNHEJ proteins among eukaryotes, the nematode Caenorhabditis elegans is unique in that it does not possess homologs of known cNHEJ processing enzymes or structural scaffolds. This raises the question of whether a basic cNHEJ system consisting only of the Ku ring and the Ligase IV ortholog LIG-4 suffices in C. elegans or whether it possesses non-conserved functional analogs of other known eukaryotic cNHEJ factors.In this thesis, I provide a definitive answer to this question by showing that nhj-1, a previously uncharacterized gene unique to the nematode order Rhabditida, is an indispensable cNHEJ factor in C. elegans. I show that some lines of the most commonly used C. elegans wild type strain, N2, exhibit unexpected stressor- and developmental stage-specific sensitivity to ionizing radiation exposure at the first larval stage (L1). This sensitivity is characterized by a high incidence of growth delay and abnormal morphological phenotypes, as well as a greatly reduced number of progeny. However, there is no evidence of germline DNA damage, which is evocative of known cNHEJ mutants in C. elegans. Using deep sequencing and CRISPR-Cas9 mutagenesis, I reveal the existence of a spontaneous nhj-1 mutation in the sensitive N2 lines, and show that IR-sensitivity arises because of the resulting nhj-1 loss of function. Furthermore, the IR-sensitivity of nhj-1 mutants is as severe as that of lig-4 or Ku ring mutants, and double mutants do not show additive IR-sensitivity, demonstrating that nhj-1 is part of the cNHEJ pathway. I also provide the first characterization of the subcellular localization of NHJ-1 and LIG-4 in the C. elegans L1 larva, the adult germline, and the adult intestine, showing a lack of complete overlap in the NHJ-1 and LIG-4 expression patterns in the L1 larva. Finally, I employ a germline cNHEJ-enabled genetic background to show that NHJ-1 likely acts downstream of Ku ring binding, at least in the context of the adult germline. My work reveals that C. elegans has restructured the ancient cNHEJ pathway to include an entirely novel regulator. It raises further questions about the regulation and mechanism of cNHEJ activity in this important model organism, and opens several research avenues in which the answers to those questions may be sought.L'intégrité physique et informationnelle de l'ADN est d'une importance capitale pour la survie, le développement et la reproduction d'organismes vivants. Parmi les diverses formes de dommages pouvant résulter des nombreuses formes intrinsèques et extrinsèques de stress génotoxique, les plus graves sont les cassures à double brin (DSB). Ces lésions sont dangereuses non seulement en raison de leur potentiel mutagène, mais également parce qu'elles perturbent la continuité physique des molécules d'ADN et risquent la perte d'un grand nombre de gènes situés en aval de la rupture. La réparation des DSBs est gérée par plusieurs voies de réparation distinctes, parmi lesquelles la classique jonction d'extrémité non homologue (cNHEJ) est la plus importante dans les tissus somatiques. Le mécanisme de cNHEJ comprend trois étapes: 1) la reconnaissance et liaison rapides de l'extrémité de l'ADN libre par la protéine hétérodimère Ku en forme d'anneau; 2) le traitement des extrémités d'ADN endommagées par un complément de nucléases, kinases, phosphatases et polymérases afin d'obtenir des extrémités compatibles avec la ligature; et 3) la ligature terminale par la ligase IV. Malgré la large conservation des protéines cNHEJ chez les eucaryotes, le nématode Caenorhabditis elegans est unique en ce qu'il ne possède aucun homologue d'enzymes de traitement de la voie cNHEJ ni d'échafaudages structuraux. Cela soulève la question de savoir si un système de cNHEJ de base composé uniquement de l'anneau Ku et de l'orthologue LIG-4 de Ligase IV suffit chez C. elegans ou s'il possède des analogues fonctionnels non conservés d'autres facteurs cNHEJ eucaryotes bien connus.Dans cette thèse, je réponds de manière définitive à cette question en montrant que nhj-1, un gène unique à l'ordre des nématodes Rhabditida et jusque-là non caractérisé, est un facteur cNHEJ indispensable chez C. elegans. Je montre que certaines lignées de la souche de type sauvage de C. elegans la plus couramment utilisée, N2, présentent une sensibilité inattendue à l'exposition aux rayonnements ionisants au premier stade larvaire (L1), qui est spécifique au type de stress et au stage de développement. Je montre que la lignée N2 sensible à l'IR présente une incidence élevée de retard de croissance et de phénotypes morphologiques anormaux, ainsi qu'un nombre considérablement réduit de descendants. Cependant, rien n'indique que l'ADN germinal ait été endommagé, ce qui évoque des mutants cNHEJ connus chez C. elegans. En utilisant le séquençage en profondeur et la mutagenèse CRISPR-Cas9, je révèle l'existence d'une mutation spontanée de nhj-1 dans les lignées sensibles de N2 et montre que cette sensibilité résulte de l'inactivation de ce gène. Je montre également que la sensibilité des mutants nhj-1 est aussi sévère que celle des mutants des anneaux lig-4 ou Ku, et que les doubles mutants ne montrent pas de sensibilité additive à l'IR, démontrant que nhj-1 fait partie de la voie de cNHEJ. Je fournis également la première caractérisation de la localisation subcellulaire de NHJ-1 et LIG-4 dans les larves de C. elegans L1, la lignée germinale adulte et l'intestin adulte, montrant un manque de chevauchement complet des modèles d'expression de NHJ-1 et LIG-4 dans les larves L1. Enfin, j'utilise un contexte génétique dans lequel la voie cNHEJ est activée dans la lignée germinale pour montrer que NHJ-1 agit probablement en aval de la liaison de l'anneau Ku, au moins dans le contexte de la lignée germinale adulte.Mon travail révèle que C. elegans a restructuré l'ancienne voie du cNHEJ pour y inclure un régulateur entièrement nouveau. Cela soulève d'autres questions sur la régulation et le mécanisme de l'activité du cNHEJ dans cet organisme modèle important et ouvre plusieurs pistes permettant de rechercher des réponses à ces questions