Tesis sobre el tema "Immunity control"

Insects are ubiquitous, diverse, and able to combat infections despite their lack of adaptive immunity. Insects have a robust innate immune system that is divided into two branches, cell-mediated and humoral. Activation of cell-mediated immune responses results in phagocytosis, nodule formation, and encapsulation by the insect’s immune cells, hemocytes. Activation of humoral immunity results in the production of anti-microbial peptides (AMPs) and phenoloxidase (PO). Insect immune responses can be plastic with development. However, research on how and why insect immunity changes with age as insects develop within a larval developmental stage (instar) is limited and contradictory. In my dissertation research, I answer two main questions: 1) how do immune responses vary within an instar and 2) what drives changes in immunity within an instar? My dissertation research showed that humoral immune responses are more robust at the beginning of the 5th and final instar in Manduca sexta (tobacco hornworm) compared to responses from animals later within that instar. Many changes occur within an instar that could affect immunity. For example, I found that protein expression of matrix metalloproteinase (MMP) in immune tissues of M. sexta decreases throughout the 5th instar. Though MMPs are involved in immune responses in other insects, MMP was not found to be immunostimulatory in M. sexta. Another important factor that changes within an instar is the level of juvenile hormone (JH). JH, a developmental hormone that prevents early molting, peaks early and decreases within an instar until molting. I determined that JH is necessary to survive an infection, control bacterial growth in hemolymph (insect blood), and mount an AMP activity immune response. My dissertation research has established that there is a development-immunity link, and that the naturally fluctuating levels of JH may mediate the effect of development on immunity.
North Dakota State University Graduate School Doctoral Dissertation Award

Three model plant and three crop plant species were grown for three generations in sand and compost. Pots were inoculated with 10 % soil initially, and with 10% of growth medium from the previous generation in generations 2 and 3, keeping replicates separate for all three generations. The microbiome community structure of the plant rhizosphere in each generation was characterised using ARISA DNA fingerprinting and 454 sequencing. Rhizosphere bacterial and fungal communities are different from those in bulk soil and there are also differences in the microbial community between different plant species. Plants both select and suppress specific bacteria and fungi in the rhizosphere microbiome, presumably via composition of their root exudates. Two out of three most abundant bacteria selected in the rhizosphere were isolated. These isolates proved to possess plant growth promotion properties. Plants are able to “farm” the soil in order to enrich it with plant growth promoting rhizobacteria (PGPR) species. However, in some plant species rhizospheres, invasions of opportunists and pathogens take place, mimicking events in plant monocultures. Other experiments using this multi-replicate system allowed for statistical analysis of the influence of Arabidopsis and Medicago mutants on the rhizosphere microbiome. Three groups of Arabidopsis mutants were tested: plants unable to produce aliphatic glucosinolates, plants impaired in the PAMP-triggered immune response and plants unable and over-expressed in methyl halides production and one group of Medicago mutants which are impaired in the mycorrhization ability. All these plant genotypes, except those for methyl-halide production and one genotype involved in PAMP response, significantly altered the rhizosphere microbiome.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
"May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1) resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune response to Salmonella infection, thereby bringing about varied outcomes in the infected host.

Thesis (Ph. D.)--West Virginia University, 2009.
Title from document title page. Document formatted into pages; contains xiv, 118 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 109-118).

The circadian clock regulates a wide range of biological processes, allowing plants to be prepared for predictable daily diurnal changes in environmental cues such as light and temperature. Recent studies have suggested that the circadian clock may also control plant immunity. The exact nature of the interaction between the circadian clock and plant pathogens remains unknown. Our focus in this study is on the elucidation of the role of the biological clock in plant immunity against the necrotrophic pathogen to Botrytis cinerea. In order to do this we tested the level of susceptibility to B. cinerea in Arabidopsis thaliana wild type and transgenic plants: toc1, cca1/lhy, cca1/toc1, lhy/toc1, cca1/lhy/toc1, GLK1 OE, GLK2 OE, glk1, glk2, and glk1/glk2. We demonstrated that the time of infection plays a role in susceptibility to B. cinerea. Specifically, we found that plants are more susceptible to infection in the subjective morning. We also found that genetic mutations in core clock components or in GLK genes leads to changes in susceptibility to B. cinerea. Our data suggests that clock genes are not solely responsible for plant immune responses to B. cinerea but rather the ways in which the biological clock system regulates outcome pathways. Furthermore, when we entrain the biological clock by changing the photoperiod (day length) in normal earth conditions LD 24h and SD 24h, we observed that short day plants had higher susceptibility to B. cinerea than long day plants. In addition, when we entrain the biological clock in different photoperiods, the LD 30h photoperiod plants displayed similar responses as those in the SD 24h photoperiod. The data indicates that day length is not responsible for the control of plant immunity; it is the ability of light to entrain the biological clock that is important. Together, the data strongly support the conclusion that the circadian clock plays a role in plant defense regulation.

Pattern recognition receptors (PRRs) are localized at the cell surface to recognize conserved microbial patterns and activate plant immunity. The activation status regulates the localization of ligand-bound PRRs and routing to late endocytic trafficking for vacuolar degradation. This ligand-induced endocytosis is conserved across PRR families, and best characterized for the receptor kinases flagellin-sensing 2 (FLS2) and EF-Tu receptor (EFR), both mediating anti-bacterial immunity. However, the molecular determinants of FLS2 and EFR endocytic trafficking, as well as the biological relevance of these processes remain poorly understood. In this study, I have dissectedlolo l the molecular code underlying ligand-mediated endocytosis. I show that flagellin induced the interaction of FLS2 with vacuolar protein sorting (VPS) 37-1, a subunit of the endosomal complex required for transport-I (ESCRT-I) that recognizes internalized ubiquitinated proteins for vacuolar sorting. This led me to investigate the role of ubiquitination in FLS2 endocytosis. Using mass-spectrometry and mutational approaches, I identified ubiquitinated FLS2 lysine residues potentially involved in FLS2 endocytic trafficking. Additionally, I revealed an involvement of the E3 ligases keep on going (KEG) and the two redundant plant U-box (PUB) 12 and PUB13 in FLS2 trafficking. I showed that PUB12/PUB13-mediated monoubiquitination of FLS2 is a key regulatory process for internalisation of activated receptors, while EFR endocytic degradation is regulated by receptor phosphorylation on the tyrosine residues 875 and 877. For both FLS2 and EFR, altering ligand-induced endocytosis did not impact the initiation of downstream signalling, demonstrating that these processes are uncoupled. Instead, my results showed that ligand-mediated endocytosis of FLS2 and EFR plays a pivotal role in maintaining chronic immune signalling responses upon long term ligand stimulation. Overall, my results uncover an important molecular mechanism regulating the subcellular trafficking of the central immune components FLS2 and EFR, and extend our understanding on how plant responsiveness to its surrounding pathogens is maintained.

As our body's guardian, the immune system maintains systemic health through removal of pathogens, damage and cancer. Ageing of the immune system is associated with compromised immune responses as well as decreased tumour surveillance and is therefore a key risk factor for major diseases in the elderly. Adaptive immune responses are mediated by T and B lymphocytes, and failure in adaptive immunity is a particular hallmark of the ageing organism. Here we show that autophagy is impaired in aged murine B lymphocytes, and loss of autophagy causes severely reduced B cell responses. Our data demonstrate that B cell senescence can be reversed in an autophagy-dependent manner by spermidine, a naturally occurring polyamine metabolite. Mechanistically, our study reveals that the translation factor eIF5A, that requires spermidine for its activation, regulates the expression of the master autophagy/lysosomal transcription factor TFEB. Importantly, we show in humans that spermidine, eIF5A and TFEB levels decrease with age and may serve as ageing biomarkers. Taken together our results indicate that the translational control of autophagy by eIF5A is dysregulated with ageing, and identify a novel pathway with therapeutic implications.

Glioblastoma multiforme (GBM) is the most lethal primary central nervous system tumor, with median survival after diagnosis of less than 12 months because dissemination into the brain parenchyma limits the long-term effectiveness of surgical resection, and because GBM cells are resistant to radiation and chemotherapy. This sad dismal prognosis for patients with GBM emphasizes the need for greater understand of the fundamental biology of the disease. Invasion is one of the major causes of treatment failure and death from glioma, because disseminated tumor cells provide the seeds for tumor recurrence. Inflammation is increasingly recognized as an important component of invasion. In the brain, inflammation can occur by activation of microglia, the resident macrophages of the brain, or by tumor-associated blood macrophages. Therefore, we hypothesize that activity of the innate immune system in the brain can influence tumor progression by secreting cytokines such as Tumor Necrosis Factor alpha (TNF-α). In this study, we show that patient-derived glioma spheres undergo morphological changes in response to TNF‑α that are associated with changes in migration behavior in vitro. These morphological changes include appearance of tumor islands in site different from where the primary tumor cells were seeded. We further showed that TNF‑α treated cells significantly increased expression of cell adhesion molecules such as CD44 and VCAM-1. Furthermore, we demonstrate increased cell density also caused increased in expression of cell adhesion molecules. The extent to which these are recapitulated in vivo will be investigated.

Preliminary experiments employing the Mudlux reporter system indicated that the ahp locus was osmotically sensitive in S. typhimurium. Studies on other bacteria supported this view. The affect of the osmotic environment of the cell upon the expression of ahp was therefore addressed in greater depth. The subsequent use of immunoblotting techniques conclusively demonstrated that chromosomal expression of the ahp locus was not affected by the osmotic environment surrounding the cell. Instead, the Mudlux element was found to alter the natural behaviour of the ahp promoter in such a way that it adopted an osmotically-regulated status, and this mode of regulation appeared to override regulation via the normal hydrogen peroxide-inducible mechanism. S. enterica is an intracellular pathogen which is capable of surviving within macrophage cells. Macrophages are equipped with an arsenal of anti-microbial effector mechanisms, including a respiratory burst which generates reactive oxygen metabolites. Since ahp had previously been shown to respond to the respiratory burst of macrophages, this study also assessed the role of oxidative stress resistance genes in the virulence of S. typhimurium. Strains of the mouse pathogen Sl1344 were constructed in which the ahp and oxyR loci were disrupted and their virulence was assessed in LD₅₀ studies. Disruption of the ahp or oxyR loci was found to have no effect upon the gross virulence of SL1344 for mice, suggesting that these loci were not essential for survival within the macrophage. The ability to develop immunity against infection by S. typhimurium is thought to correlate with the development of immunity against bacterial antigens which are expressed in vivo. As a further part to this study, the immunological responses of mice to two S. typhimurium-derived polypeptides, AhpC and GroEL, following cloning and overexpression of these proteins, were examined. Mice previously infected with an attenuated strain of S. typhimurium were shown to elicit significant delayed-type hypersensitivity reactions following subcutaneous injection of these polypeptides 33 and 104 days post-infection. Moreover, AhpC-and GroEL-specific antibodies were detected during the course of infection of mice with S. typhimurium.

La méthylation et déméthylation de l'ADN jouent un rôle majeur dans la stabilité des génomes, l'empreinte génomique, la paramutation et le développement. En revanche, le rôle de cette régulation épigénétique a été peu étudiée dans les interactions hôtes-pathogènes. Dans ce projet de thèse, nous avons tout d'abord montré que la méthylation de l'ADN régule négativement la résistance d'Arabidopsis thaliana à une souche de Pseudomonas syringae pathogène. Nous avons également identifié un grand nombre de gènes de l'immunité ciblés directement par la méthylation de l'ADN dirigée par petits ARN dans leurs régions promotrices. Nous proposons que cette régulation génique permettrait de maintenir une faible expression basale de ces gènes et d'éviter ainsi des effets délétères qui seraient causés par une expression constitutive de la réponse immunitaire. De plus, nous montrons que la déméthylase active REPRESSOR OF SILENCING 1 (ROS1) facilite l'activation transcriptionnelle de gènes de l'immunité en laissant potentiellement des éléments de régulation en cis accessibles à des facteurs de transcription. Nous avons également démontré que ce facteur contribue à la résistance à P. syringae chez Arabidopsis, caractérisant ainsi la première déméthylase eucaryote dans la résistance antibactérienne. Sur la base de ces résultats, nous proposons que la méthylation de l'ADN maintient une faible expression basale de gènes de l'immunité en absence de pathogène, tandis que la déméthylation active assure une induction rapide de ces gènes au cours de la réponse immunitaire en favorisant potentiellement le recrutement de facteurs de transcription sur la chromatine
DNA methylation and demethylation are regulatory processes involved in genome stability, genomic imprinting, paramutation and development. Until recently, very little was known about the role of these epigenetic processes in plant disease resistance and in the transcriptional control of immune-responsive genes. Here we provide evidence that DNA methylation negatively regulates antibacterial resistance against a virulent Pseudomonas syringae strain in Arabidopsis. Accordingly, we have identified a subset of defense genes that are targeted and repressed by RNA-directed DNA methylation (RdDM), presumably to prevent trade-off effects that would be caused by their constitutive expression and/or sustained induction. In addition, we found that the active DNA demethylase facilitates the transcriptional activation of some of these defense genes by pruning DNA methylation at their promoter regions and leaving cis-elements accessible for transcription factor binding. In addition, we show that the active demethylase REPRESSOR OF SILENCING 1 (ROS1) positively regulates late immune responses including Pathogen Associated Molecular Pattern (PAMP)-triggered callose deposition and salicylic acid (SA)-dependent defense response. We also demonstrate that ROS1 restricts Pto DC3000 propagation in Arabidopsis leaf secondary veins, providing the first example for a role of an active DNA demethylase in antibacterial resistance. Based on these findings we propose that DNA methylation maintains a low basal expression of some immune-responsive genes in normal growth condition, while active DNA demethylation ensures a rapid and pervasive induction of these genes upon bacterial pathogen detection

Poly(ADP-ribose) polymerases belong to a family of proteins with 17 members in human beings. PARP1, the founding member of the family is a protein that synthesizes linear or branched polymers of ADP-ribose on itself or on target proteins. Different members of this family, that do not all possess ADP-ribosyl polymerase activity, are involved in the regulation of various cellular mechanisms. Some members of the family are particularly involved in the positive or negative control of the immune response. PARP1 is a key player in the regulation of inflammation, through its positive control of cell death and of proinflammatory cytokine production. On the other hand, the tankyrases (PARP5a and PARP5b) and PARP14 seem to regulate inflammatory responses in a negative fashion. PARP12 is a poorly characterized member of the family, whose expression is greatly increase following stimulation with type-I interferons, cytokines mainly involved in antiviral defences.

PARP12 is a protein that possesses three main domains: A putative RNA binding N-terminal domain composed of tandem CCCH zinc-fingers, a central WWE domain and a C-terminal PARP catalytic domain. In this work, we have shown that the expression of PARP12 is strictly-dependent on type-I interferons, that it possesses ADP-ribosyl transferase activity and that in can regulate the translation of messenger RNA into proteins. PARP12 can be found in stress granules, sites of storage of untranslated mRNAs, and is capable of directly inhibiting the translation of a reporter mRNA when tethered to it, in a manner dependent on its catalytic activity. Furthermore overexpression of wild-type PARP12, in contrast to overexpression of a mutant with no detectable catalytic activity (PARP12-G575W), leads to a general arrest of most cellular translation.

On the other hand, we have shown that PARP12 can activate the transcription of genes under the control of an NFκB-dependent promoter, especially when its zinc-fingers are deleted or mutated (PARP12ΔZnF). PARP12ΔZnF is located in structures that can enclose TRIF, RIP1, NEMO, p62/SQSTM1 and ubiquitin. These proteins have all possess an important role in the activation of NFκB signalling cascades. Moreover, we have shown that endogenous PARP12 is situated in ALIS (Aggresome-Like Induced Structures) in LPS-stimulated macrophages. These structures have a possible role in the presentation of antigens on class I major histocompatibility complexes, implying that PARP12 may be involved in the regulation of antigen presentation.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

This thesis examined the molecular mechanisms that are at the basis of parasite-host interactions during malaria infection, specifically focussing on antibody function and interactions during inflammation. Plasmodium parasites were recently discovered to grow slower during acute infection. This project built on that evidence and identified a role for inflammation as well as detected an initial response by the parasite to inflammation.

CD8+ T cells are essential for host protection against intracellular pathogens and tumors. During antigen-driven responses, CD8+ T cell fate is governed by transcriptional and epigenetic processes that allow naïve CD8+ T cells to develop into a wide range of effector and conventional memory cell subsets. Over the last decades, novel techniques and major efforts led to a better understanding of the origin, nature, and short- and long-term effects of these processes on individual CD8+ T cells. Under certain conditions, naïve CD8+ T cells can acquire memory phenotype and functions in an antigen-independent manner. Although homeostatic cytokines and initial activation pathways that drive the development of these unconventional memory cells had been identified, the ensuing transcriptional profile of these cells and their degree of similarity with conventional memory cells remained ill-defined. The epigenetic events that accompany unconventional memory formation were also not known.Here, we show that innate memory cells, a type of thymic unconventional memory cells, are transcriptionally close to conventional memory cells but only partially epigenetically programmed toward the full memory fate. We also show that the sole overexpression of the transcription factor Eomesodermin (EOMES), a master regulator of effector and conventional memory cells, is able to drive many of the phenotypical, functional, transcriptional, and epigenetic features of innate memory cells, and to induce the recruitment of BRG1, a member of chromatin remodeling complexes, to innate memory gene regulatory regions. We further show that the in vivo interleukine-4-dependent development of innate memory cells is largely dependent on BRG1. We bring to light that, in innate memory cells, EOMES is recruited in many instances to genomic regions previously bound by the transcription factor RUNX3. Overall, we provide insights into the mechanisms that allow memory cell formation and T cell receptor stimulation to be uncoupled.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

El presente trabajo tiene por objeto alcanzar un modo de comprender la ineficacia del Derecho penal frente a la corrupción pública con fines de orden patrimonial. La comprensión gnoseológica de tal problemática es, fundamentalmente, de naturaleza criminológica. Y se dice «un modo» de comprensión, puesto que el paradigma epistemológico, la perspectiva teórica y la postura metodológica que se adopta para lograr tal cometido, permiten establecer tan sólo un «enunciado de posibilidad» sobre la definición del problema referenciado. El enfoque analítico, por tanto, se define por la formulación de una base gnoseológica que permite representar parte de las razones de tal ineficacia. Para ello se asume una perspectiva analítica desde la que concibe el comportamiento delictivo no tanto en su configuración externa y material (objetiva), sino en su dimensión interna y significativa (subjetiva). Planteado así el problema de estudio y el enfoque analítico, la cuestión central a resolver se define por el conjunto de factores que facilitan la impunidad del fenómeno criminal analizado. El fin último de la identificación de las circunstancias generadoras de impunidad se concreta de este modo en la promoción político-criminal de una línea alternativa de prevención que, acorde con la mirada subjetiva del fenómeno estudiado, no se identifica llanamente con las estrategias de intervención situacional del delito, sino con la redefinición de la posición social e institucional frente al interés jurídico tutelado por el Derecho penal. Para alcanzar semejantes propósitos, como se proyecta en la amplia Introducción General que se plantea, se propone una ruta de análisis que bascula entre el acercamiento interdisciplinar a los factores que generan la impunidad del fenómeno criminal y la proposición continua de una serie de alternativas de intervención que van desde la profusión moral que se halla tras el interés superior protegido por la norma jurídico-penal, hasta la estructura misma de las figuras dogmáticas que dificultan su función preventivo-general. Para ese recorrido se establece una categoría central vinculada, precisamente, a esa aspiración de prevención: la función intimidatoria del Derecho penal. Alrededor de esta categoría se desarrolla el problema de análisis. El objetivo general bajo el que se vehicula tal estudio, concretamente, se dirige a establecer por qué la pena no disuade. Y aquí es donde adquiere su mayor importancia la mirada subjetiva adoptada. Para responder a semejante cuestión se propone, con la idea de comprender cuál es el origen y la naturaleza de las justificaciones a las que apela el delincuente corrupto para «desviarse» del curso «correcto» ordenado por el Derecho, un adentramiento al mundo de significados del propio infractor. Y al tratarse de un intento de comprensión comportamental desde la propia mirada del actor criminal –histórica y contextualmente situado-, ineludiblemente se parte de un conjunto de planteamientos de naturaleza psicológica y sociológica que, reunidos, permiten formar una propuesta criminológica desde la que no sólo se alcanza un modo de comprensión del fenómeno objeto de punición, sino que también permite proponer una redefinición de la manera tradicional de abordar el estudio del delito y la promoción de políticas de prevención.
This paper aims at reaching a way of understanding the ineffectiveness of the criminal law against the public corruption regarding the purpose of financial gain. The gnoseological understanding of this problem is fundamentally of a criminological nature. And it is said «a way» of understanding, given that the epistemological paradigm, the theoretical perspective and the adopted methodology used to achieve such a purpose, allow to establish just a «statement of possibility» regarding the definition of the highlighted issue. The analytical approach, therefore, is defined by the formulation of a gnoseological framework that allows to represent part of the reasons for such ineffectiveness. In order to do so, an analytical perspective is adopted from which the criminal behavior is conceived not so much from its external and material configuration (objective), but from its internal and significant dimension (subjective). Raised are the statement's problem and the analytical approach, the central issue to resolve is defined by a set of factors that facilitate the impunity of the analyzed criminal phenomenon. The ultimate purpose of the identification of the circumstances generating impunity is materialized from the political- criminal promotion of an alternative line of prevention that, in accordance with the subjective analysis of the studied phenomenon, is not quite simply identified with the crime's situational intervention strategies, but with the redefinition of the social and institutional position in front of the legal interest protected by the Criminal Law. To achieve similar purposes, as projected in the broad General Introduction which proposes a path analysis scale that moves between the interdisciplinary approach to the factors that cause the impunity of the criminal phenomenon and the continuing proposition of a series of intervention alternatives ranging from the abundance of moral principles found behind the superior interests protected by the criminal-legal legislation up to the structure of the dogmatic figures that hinder its general-preventive function. To carry out this analysis, it is established that a central category linked precisely to the aspiration of prevention: the intimidatory role of Criminal Law. Around this category the problem of analysis is developed. The overall objective under which such a study is conveyed, specifically is aimed to establish why the punishment does not dissuade the offenders. And here is where the adopted subjective approach acquires its greatest importance. To respond to such an issue, it is proposed along with the idea of understanding the origin and nature of the justifications that the corrupt offender appeals to in order «deviate» from the «correct» course ordered by the Law, a deeper analysis into the world of meanings from the very own offender. And being an attempt of understanding the behavior from the very own offender's view –historical and contextually located-, undoubtedly it begins from a set of approaches of psychological and sociological nature that together, enable to formulate a criminological proposal which not only achieves a way of understanding the phenomenon subject to punishment, but it also proposes a redefinition of the traditional way of dealing with the study of the crime as well as promoting preventive policies.
El present treball té per objecte aconseguir una manera de comprendre la ineficàcia del Dret penal enfront de la corrupció pública amb finalitats d'ordre patrimonial. La comprensió, en un sentit gnoseològic, de tal problemàtica és, fonamentalment, de naturalesa criminològica. I es diu «una manera» de comprensió, ja que el paradigma epistemològic, la perspectiva teòrica i la postura metodològica que s'adopta per assolir tal comesa, permeten establir tan sols un «enunciat de possibilitat» sobre la definició del problema esmentat. L'enfocament analític, per tant, es defineix per la formulació d'una base gnomologia que permet representar part de les raons de tal ineficàcia. Per a això s'assumeix una perspectiva analítica des de la qual concep el comportament delictiu no tant en la seva configuració externa i material (objectiva), sinó en la seva dimensió interna i significativa (subjectiva). Plantejat així el problema d'estudi i l'enfocament analític, la qüestió central a resoldre es defineix pel conjunt de factors que faciliten la impunitat del fenomen criminal analitzat. La fi última de la identificació de les circumstàncies generadores d'impunitat es concreta d'aquesta manera en la promoció polític-criminal d'una línia alternativa de prevenció que, d'acord amb la mirada subjectiva del fenomen estudiat, no s'identifica planament amb les estratègies d'intervenció situacional del delicte, sinó amb la redefinició de la posició social i institucional enfront de l'interès jurídic tutelat pel Dret penal. Per aconseguir semblants propòsits, com es projecta en l'àmplia Introducció General que es planteja, es proposa una ruta d'anàlisi que bascula entre l'acostament interdisciplinari als factors que generen la impunitat del fenomen criminal i la proposició contínua d'una sèrie d'alternatives d'intervenció que van des de la profusió moral que es troba després de l'interès superior protegit per la norma jurídic-penal, fins a l'estructura mateixa de les figures dogmàtiques que dificulten la seva funció preventiu-general. Per a aquest recorregut s'estableix una categoria central vinculada, precisament, a aquesta aspiració de prevenció: la funció intimidatoria del Dret penal. Al voltant d'aquesta categoria es desenvolupa el problema d'anàlisi. L'objectiu general sota el qual es vehicula tal estudi, concretament, es dirigeix a establir per què la pena no dissuadeix. I aquí és on adquireix la seva major importància la mirada subjectiva adoptada. Per respondre a semblant qüestió es proposa, amb la idea de comprendre quin és l'origen i la naturalesa de les justificacions a les quals apel·la el delinqüent corrupte per «desviar-se» del curs «correcte» ordenat pel Dret, un endinsament al món de significats del propi infractor. I en tractar-se d'un intent de comprensió del comportament des de la pròpia mirada de l'actor criminal –històrica i contextualment situat-, ineludiblement es parteix d'un conjunt de plantejaments de naturalesa psicològica i sociològica que, reunits, permeten formar una proposta criminològica des de la qual no només s'aconsegueix una manera de comprensió del fenomen objecte de punició, sinó que també permet proposar una redefinició de la manera tradicional d'abordar l'estudi del delicte i la promoció de polítiques de prevenció.

Thesis advisor: Ellen Winner
Cognitive control, the ability to limit attention to goal-relevant information, subserves higher-order cognitive functions such as reasoning, attention, planning and organization. Counterintuitively, deficits in these functions have proven advantageous in certain contexts: low cognitive control means less filtering of attention, and such unfiltered attention leads to novel solutions in insight problem solving contexts. Insight is the clear and often sudden discernment of a solution to a problem by means that are not obvious, and it plays an indispensable role in creative thinking. This study examined whether insight problem solving is a compensatory advantage for children of low socioeconomic status because of their known deficits in cognitive control. One hundred and forty-eight children ages 4 to 11 years old, each completed two insight problem solving tasks (the Box Problem and the Pencil Problem) and a cognitive control task (the Flanker/Reverse Flanker). In addition, their parents completed a sociodemographic questionnaire, which was used as a measure of their socioeconomic status and child rearing values of obedience versus independence. No association was found between children’s socioeconomic status and their ability to use insight to solve a problem. Results did show that older children exhibited less cognitive flexibility than did to younger children, and that diminished cognitive flexibility correlated with older children’s ability to solve the Box Problem; however, this effect did not hold when age, sex, race, socioeconomic status, and parental report of obedience versus independence, were accounted for. Ultimately, age was the only significant predictor of children’s insight problem solving ability, such that older children were significantly more likely to solve the Box Problem and to arrive at a solution more quickly for the Pencil Problem compared to younger children. Findings from this study are explained using evidence from research on children’s tool innovation showing that young children are poor at inventing tools, and that older children’s ability to use objects for atypical functions may be the result of their greater exposure to and experience with tools
Thesis (PhD) — Boston College, 2021
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Psychology

The human immunodeficiency virus (HIV) epidemic is far from over. Antiretroviral therapy (ART) is effective at suppressing viral replication within a patient but it must be taken daily and is life-long. Therefore, the development of a therapy that could induce drug-free remission or constitute a functional cure is a key focus of HIV research. In this thesis I explore three mechanisms which could lead to more individuals being able to control their viraemia in the absence of ART: (1) T-cell immunity, (2) early initiation of ART, and (3) viral evolution. Firstly, a strong HIV-specific T-cell response has been linked to rare cases of spontaneous viral control, but the extent to which this arm of the immune response contributes to viral control is debated. Several types of data are used to answer this question, including the rate at which the virus evolves to escape the CD8+ T-cell response. I study the frequency of incident immune escape in the largest cohort used for this purpose to date. Secondly, some patients, with characteristics dissimilar to spontaneous HIV controllers, are able to control the virus for years after the interruption of ART that was initiated early in infection. I use mathematical models to investigate a new hypothesis for the differing outcomes of early- and late- initiated ART. Thirdly, since HIV is a relatively new infection of humans it is still adapting to its new host. Recent studies suggest that the virus could be evolving towards decreased virulence at the population level. I study whether the widespread administration of ART has the potential to alter the course of virulence evolution and might result in a further attenuated virus. I conclude by discussing the implications of these results for viral control at the individual level and also for population level epidemic control.

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

La thèse vise à réfléchir, à partir d'une approche foucaldienne, à la signification de la sécurité dans le monde occidental contemporain. Pour ce faire, une analyse socio-technique précise d'un dispositif de sécurité est réalisée. L'objet en question est la caméra de vidéosurveillance et son perfectionnement intelligent. En interrogeant les modalités d'enregistrement et de traitement de l'image, dans l'univers numérique, nous interrogeons la place de l'humain au sein de la gouvernementalité ainsi que les formes de subjectivation qui en résultent. Pour l'intelligibilité du propos, nous retraçons les différents modes de gouvernement ainsi que les formes principales de contrôle relevés par Michel Foucault avant de proposer une actualisation du mode de gouvernementalité biopolitique à la lueur des dispositifs de sécurité étudiés
The thesis aims to examine, from the perspective of Foucault's work, the meaning of security in the contemporary occidental world. To do this, a precise socio-technical analysis of a security device has been conducted. The device itself is the CCTV camera and its continuous smartness improvements. By questionning the ways of image recording and processing, in the digital environment, we are putting into question the man's place into governmentality as well as its kinds of subjectivity. For a better understanding, we trace the various governing modes and the main forms of control pointed out by Michel Foucault before submitting an updated biopolitical governing mode in the light of the examined security devices

Bovine mastitis is one of the most significant causes of economic losses for the dairy industry. On the other hand, public health authorities advise prudent use of antibiotics because they could promote bacterial resistance and leave residues in food chain. The dairy industry could benefit from the development of safe antimicrobial agents and bacteriocins could be attractive alternatives to antibiotics. Due to the safety of Lactic acid bacteria (LAB), their bacteriocins have the potential to be used as antimicrobials in veterinary clinical application. We analyzed the efficacy of antibacterial substances produced by bacteriocinogenic Lactococcus lactis subsp. lactis strains against contagious and environmental mastitis pathogens. Thereafter, we investigated how lactococcal strains or their bacteriocins could influence mammary gland innate immune response in vitro. Out of 65 LAB strains tested, 3 were active against mastitis pathogens: 2 strains produced Nisin, one Lacticin 481 and in addition a novel molecule with likely antibacterial activity. To analyze the immune response of mammary epithelial cells when stimulated with lactococcal strains or bacteriocins, a stabilized epithelial cell line, BME-UV1, was used. Both lactococcal live cultures and their antibacterial products were shown to modulate the non-specific immune response of BME-UV1 cells: Lysozyme and N-acetil-β-D-glucosaminidase excretion were overall enhanced by bacteriocins and live-culture treatments, while intracellular amounts were unaffected by treatments. Proinflammatory cytokine expression of treated BME-UV1 was similar to that observed in control cells, except for Lactococcus lactis subsp. lactis SL153. Such strain induced a significant reduction of TNFα transcriptional level. The stimulation of enzyme secretion due to the administration of lactococci or of their antibacterial products, with potential enhancement of pathogens cleaning, can be of interest for the prevention of intra mammary infections. In addition, Lactococcus lactis subsp. lactis SL153 strain could be advantageous for its potential anti-inflammatory properties and could be of interest for the development of intra-mammary probiotic treatments.

Факториальные числа находят практическое применение при генерации перестановок. Перестановки в свою очередь широко используются для решения задач комбинаторной оптимизации, например, задач поиска оптимального решения, а также при помехоустойчивой передаче данных и защите их от несанкционированного доступа.

The biological control of plant pests with beneficial microbes has become increasingly important over the last decades. Soil microbes such as fungi and bacteria colonise the roots of plants and promote their growth. Some beneficial microbes can trigger a weak plant defence response that enhances the immune response of the plant at subsequent pathogen attacks and therefore increase the resistance of the plant to other invaders. This mechanism is called “priming”. While biocontrol agents are applied against a variety of plant pests fundamental knowledge of the molecular mechanisms of plant-microbe interactions is still lacking. Especially molecular studies on the role of resistance genes in the interaction of plants with beneficial endophytic fungi are rare. In this study it was investigated how the fungal biocontrol agent Acremonium alternatum affects the development of the clubroot pathogen Plasmodiophora brassicae within the plant host Arabidopsis thaliana. Clubroot is a devastating disease in crop plants such as cabbage and rapeseed and causes abnormal root growth that leads to so called “club roots”. P. brassicae develops within the plant roots and forms resting spores that are very durable and stay infective in soils for up to 2 decades. The control of clubroot by chemical means is difficult and the disease continues to spread on all continents and was also found in Saxony, Germany in recent years. In 2 preliminary studies the co-inoculation of clubroot plants with the fungus A. alternatum resulted in reduced clubroot symptoms in Chinese cabbage and Arabidopsis. It was therefore hypothesised that A. alternatum induces resistance mechanisms in the plant and thus enhances immunity. The focus of this study was to test this hypothesis by carrying out expression analyses on root tissue of infected Arabidopsis plants. For this the plants were inoculated with spores of P. brassicae and A. alternatum before RNA was extracted from the roots, followed by cDNA synthesis and quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). A microarray of root tissue of infected Arabidopsis plants was carried out to depict the events at the stage of initial root hair infection with the clubroot pathogen. The findings from the gene expression analyses were verified for 2 genes with Arabidopsis mutants that are defective in the respective gene and with 2 overexpressor lines. Clubroot symptoms were assessed by rating the root galls according to their stage of development. The overall plant health was further evaluated by recording the developmental stage of the plants (generative vs. vegetative), stem lengths and plant biomass. In addition, 2 local varieties of the economically important crop plant rapeseed (Brassica napus var. Ability and var. Visby) were investigated with qRT-PCR and by recording the disease parameters just described. A second goal of this study was to assess the general biocontrol potential of the yet relatively unknown endophyte A. alternatum in terms of enzymatic activity and competitive behaviour against other phytopathogenic fungi. The potential of this fungus for the use in integrative pest management was investigated. The results presented here are novel findings for this fungus and have not been studied before. The microarray from Arabidopsis roots revealed that the clubroot pathogen P. brassicae suppresses its recognition by pathogen receptors of the plant and thus prevents the host to induce resistance mechanisms. The fungus A. alternatum boosted the level of the pathogen recognition-related genes BAK1 and FLS2 and thus helped to establish early plant defence responses. PCR analyses confirmed that these early responses led to salicylic acid-dependent resistance in the plants which was maintained for several days as shown by elevated levels of the PATHOGENESIS-RELATED gene PR1. Marker genes for an alternative resistance pathway that is mediated over the plant signals jasmonate and ethylene were not activated in Arabidopsis. The co-inoculation of Arabidopsis plants with the endophyte A. alternatum resulted in a significant reduction of clubroot symptoms by up to 24%. In rapeseed the reduction of disease symptoms was 19% and 28% when the plants were treated with a crude cell wall extract of A. alternatum before inoculation with the clubroot pathogen. PCR analyses from Arabidopsis showed a strong response of pathogen recognition genes to the cell wall extract and spores of the endophytic fungus. In rapeseed all of the investigated pathogen recognition genes were upregulated after the endophyte treatment but not with the clubroot pathogen. Together with the PCR results from the microarray these findings suggest that A. alternatum primes its host plant and enhances the resistance of the plant towards P. brassicae. In addition, the fungus increased biomass, stem lengths and survival rates of clubroot-infected plants. In vitro tests revealed that the endophyte can solubilise phosphate and is not very competitive against other phytopathogenic fungi such as Aspergillus or Fusarium which is likely an effect of the relatively slow growth of the endophyte on agar plates. From this study it can be concluded that i) the fungus Acremonium alternatum induces resistance mechanisms in Arabidopsis and 2 Brassica napus cultivars and facilitates the recognition of the clubroot pathogen Plasmodiophora brassicae; ii) that Arabidopsis and Brassica react differently to this beneficial microbe, a fact that has been observed for Plasmodiophora and other microorganisms as well; iii) living spores are not necessary for clubroot biocontrol in rapeseed as a crude cell wall extract reduces symptoms more efficiently. Overall the endophyte A. alternatum is a very promising candidate for the use in integrative pest management in plant strengtheners or as biocontrol agent
Die biologische Kontrolle von Pflanzenkrankheiten gewinnt zunehmend an Bedeutung. Bodenbewohnende Mikroben wie Pilze oder Bakterien kolonisieren die Wurzeln von Pflanzen und fördern deren Wachstum. Einige dieser förderlichen Mikroben aktivieren eine schwache Abwehrreaktion in der Pflanze die sich verstärkt bei einer weiteren Infektion mit einem Krankheitserreger. Dieser Mechanismus, den man “Priming” nennt, führt zu einer verbesserten Resistenz der Pflanze gegenüber Pflanzenpathogenen. Obwohl natürliche Schädlingsbekämpfer bereits gegen eine Vielzahl an Krankheiten eingesetzt werden, weiss man über grundsätzliche molekulare Mechanismen dieser Pflanzen-Mikroben-Interaktionen nur wenig. Besonders die Rolle von Resistenzgenen ist bisher wenig erforscht, welche bei der Beziehung zwischen Pilzen und Pflanzen eine Rolle spielen. In der hier vorliegenden Arbeit wurde untersucht, wie der endophytische Pilz Acremonium alternatum die Entwicklung des Krankheitserregers Plasmodiophora brassicae in der Pflanze Arabidopsis thaliana beeinflusst. Die Kohlhernie, ausgelöst von P. brassicae, ist eine verheerende Krankheit die u. a. bei Kohl und Raps auftritt und Wurzelgallen, so genannte “Hernien”, hervorruft. Der Krankheitserreger entwickelt sich im Wurzelsystem der Pflanze und bildet Dauersporen, die bis zu 20 Jahre lang im Boden infektiös überdauern können. Ein Eindämmen der Krankheit mit Pflanzenschutzmitteln ist durch den komplexen Lebenslauf des Erregers sehr schwierig, das führte zu einer weltweiten Verbreitung der Kohlhernie. Auch in Sachsen wurden in den letzten Jahren Fälle von Kohlhernie gemeldet. Wie 2 Studien zeigen, führt die Ko-Inokulation von Kohlhernie-erkrankten Pflanzen mit A. alternatum zu einer Verringerung der Symptome in Chinakohl und Arabidopsis. Es wurde daher die Hypothese aufgestellt, dass der Pilz Resistenzmechanismen in der Pflanze anschaltet und damit ihre Immunität erhöht. Um diese Hypothese zu testen, wurden in der hier vorliegenden Studie Genexpressionsanalysen an infizierten Arabidopsiswurzeln durchgeführt. Dafür wurden die Pflanzen zunächst mit Sporen des Kohlhernieerregers und des Pilzes inokuliert, es wurde RNA aus den Wurzeln extrahiert, in cDNA umgeschrieben und diese mittels quantitativer Reverse-Transkriptase-Polymerasenkettenreaktion (RT-qPCR) untersucht. Ein Microarray von Wurzeln infizierter Pflanzen wurde durchgeführt um die Ereignisse abzubilden, die sich zeitnah nach der Infektion in den Wurzeln abspielen. Die Ergebnisse der Genexpressionsanalysen wurden dann an Arabidopsismutanten, die einen Gendefekt im jeweiligen Gen haben, und an Überexprimierer-Pflanzen verifiziert. Kohlherniesymptome an Pflanzen wurden durch eine Kategorisierung der Schadsymptome erfasst. Die allgemeine Pflanzengesundheit sowie der Entwicklungsstand der Pflanze, Stengellängen und das Frischgewicht wurden bestimmt. Zusätzlich wurden 2 Rapssorten, die in Sachsen angebaut werden, untersucht im Hinblick auf die Krankheitsenwicklung und die Reguation von Abwehrgenen. Ein weiteres Ziel dieser Arbeit war es das Biokontrollpotential des bisher schlecht untersuchten Pilzes A. alternatum zu bestimmen. Dazu wurde in vitro die Enzymaktivität des Pilzes getestet sowie seine Konkurrenzfähigkeit gegenüber anderen pflanzenpathogenen Pilzen. Das Potential des Pilzes für die Anwendung im integrierten Pflanzenschutz wurde getestet. Die hier präsentieren Ergebnisse stellen neue Erkenntnisse dar, die für diesen Pilz noch nie untersucht wurden. Der Microarray von Arabidopsiswurzeln zeigte, dass der Kohlhernieerregers die Erkennung durch die Pflanze verhindert und damit Abwehrmechanismen verhindert. Der Pilz A. alternatum förderte die Aktivität der pflanzlichen Erkennungsrezeptoren FLS2 und BAK1 und setzte damit die Erkennung von P. brassicae in Gang. PCR-Analysen ergaben, dass diese früh induzierten Abwehrmechanismen zu einer systemischen Resistenz in der Pflanze führte durch die Aktivierung des Pathogenese-relevanten Gens PR1. Genmarker, die die Aktivität eines alternativen, von Jasmonat und Ethylen vermittelten Abwehrweges anzeigen, waren nicht ativiert. Die Ko-Inokulation von Arabidopsis mit dem Endophyten führte zu einer signifikanten Reduktion der Krankheitssymptome um 24%. In Raps betrug die Reduktion 19% und 24% wenn die Pflanzen vor der Kohlhernie-Infektion mit einem Zellwandextrakt des Pilzes behandelt wurden. Mittels PCR konnte gezeigt werden, dass Gene für das Erkennen von Pathogenen in der Wurzel von Arabidopsis auf den Zellwandextrakt und Sporen des Pilzes reagieren. In Raps wurden alle der untersuchten Erkennungsgene aufreguliert nach der Infektion mit A. alternatum, nicht jedoch bei der Infektion mit P. brassicae. Zusammenfassend lässt sich sagen, dass der endophytische Pilz A. alternatum die Wirtspflanze auf eine folgende Infektion vorbereitet (Priming) und systemische Abwehr-mechanismen in der Pflanze induziert, wenn diese mit Kohlhernie infiziert ist. Außerdem treibt der Pilz das Sprosswachstum voran, erhöht die Biomasse und fördert das Überleben von Kohlhernie-infizierten Pflanzen. In vitro-Tests ergaben, dass der Endophyt Kalziumphosphat löslich machen kann und wenig kompetitiv gegenüber Pflanzenpathogenen wie Aspergillus oder Fusarium ist. Dies ist vermutlich mit dem langsameren Wachstum des Endophyten im Gegensatz zu den anderen Pilzen zu erklären. Aus den Ergebnissen dieser Arbeit lassen sich folgende Schlüsse ziehen: i) der endophytische Pilz Acremonium alternatum induziert Resistenzmechanismen in Arabidopsis und Raps und und fördert die Erkennung des Kohlhernieerregers Plasmodiophora brassicae; ii) Arabidopsis und Raps reagieren unterschiedlich auf diesen förderlichen Pilz, ein solcher Unterschied wurde bereits für Plasmodiophora und andere Mikroben beschrieben; iii) lebende Sporen des Pilzes sind nicht notwendig um Krankheitssymptome der Kohlhernie in Raps zu verringern, ein Zellwandextrakt von A. alternatum ist dafür besser geeignet. Ganz allgemein lässt sich sagen, dass der endophytische Pilz Acremonium alternatum ein sehr vielversprechender Kandidat ist für den Einsatz im integrierten Pflanzenschutz in Pflanzenstärkungsmitteln oder als Biokontrollorganismus

The porcine proliferative enteropathies (PPE) are a group of diseases ranging from intestinal adenomatosis (PIA), a chronic condition causing reduced growth rates in post weaning pigs, to the often fatal proliferative haemorrhagic enteropathy (PHE), resulting in intestinal haemorrhage. PHE predominantly occurs in older and heavier pigs than the chronic disease PIA. This thesis examined whether the age when susceptible pigs are infected affects the clinical response to L.intracellularis infection. The characteristic pathologic lesion of PPE is the abnormal proliferation of crypt epithelial cells in the ileum and colon. Closely associated with this proliferation is the presence of an obligately intracellular bacterium, Lawsonia intracellularis. Characterisation of L.intracellularis was performed in in-vitro co-cultures of L.intracellularis extracted from PHE-affected mucosa. The efficacy of antimicrobials to inhibit the growth of L.intracellularis in-vitro was evaluated and compared with isolates cultured in the United Kingdom. The results were analysed with respect to medication strategies currently used to control PPE in piggeries. PPE occurs in virtually all piggery management systems, including newly developed systems that are aimed at improving the herd health, such as segregated early weaning and multiple site production. PPE is currently controlled in Australia with the routine addition of antimicrobials in pig feed, in particular olaquindox. Recommendations to reduce the use of feed-based antibiotics in Australia require the development of alternate strategies to control diseases such as PPE. Sequential outbreaks of PHE reported in minimal disease herds suggested that pigs could develop immunity to disease. An experimental model of L.intracellularis infection was developed in this thesis to demonstrate that immunity to re-infection with L.intracellularis could be developed. Infection was monitored by detection of faecal shedding of L.intracellularis and serum IgG antibodies against L.intracellularis. Two in-feed antimicrobial strategies were analysed in this thesis for their ability to induce the development of immunity to L.intracellularis, while avoiding clinical signs of disease. The first strategy evaluated the use of low levels of in-feed antimicrobials to allow subclinical infection and the development of immunity. The second strategy evaluated the use of high levels of in-feed antimicrobials to terminate infection two weeks after exposure to L.intracellularis. Gaining a greater understanding of how L.intracellularis infection is spread both within and between piggeries will enable the development of management strategies to control the spread of infection. This thesis examined the possibility that other species in contact with pigs and piggeries such as rats, mice and birds may transmit infection to pigs. The transmission of infection between pigs via the faecal/oral route was also examined, as was the survival and infectivity of L.intracellularis over time. Ultimately this thesis aimed to understand the pattern of L.intracellularis infection and the survival and transmission of L.intracellularis in order to develop effective control measures for PPE, especially in minimal disease herds.

The porcine proliferative enteropathies (PPE) are a group of diseases ranging from intestinal adenomatosis (PIA), a chronic condition causing reduced growth rates in post weaning pigs, to the often fatal proliferative haemorrhagic enteropathy (PHE), resulting in intestinal haemorrhage. PHE predominantly occurs in older and heavier pigs than the chronic disease PIA. This thesis examined whether the age when susceptible pigs are infected affects the clinical response to L.intracellularis infection. The characteristic pathologic lesion of PPE is the abnormal proliferation of crypt epithelial cells in the ileum and colon. Closely associated with this proliferation is the presence of an obligately intracellular bacterium, Lawsonia intracellularis. Characterisation of L.intracellularis was performed in in-vitro co-cultures of L.intracellularis extracted from PHE-affected mucosa. The efficacy of antimicrobials to inhibit the growth of L.intracellularis in-vitro was evaluated and compared with isolates cultured in the United Kingdom. The results were analysed with respect to medication strategies currently used to control PPE in piggeries. PPE occurs in virtually all piggery management systems, including newly developed systems that are aimed at improving the herd health, such as segregated early weaning and multiple site production. PPE is currently controlled in Australia with the routine addition of antimicrobials in pig feed, in particular olaquindox. Recommendations to reduce the use of feed-based antibiotics in Australia require the development of alternate strategies to control diseases such as PPE. Sequential outbreaks of PHE reported in minimal disease herds suggested that pigs could develop immunity to disease. An experimental model of L.intracellularis infection was developed in this thesis to demonstrate that immunity to re-infection with L.intracellularis could be developed. Infection was monitored by detection of faecal shedding of L.intracellularis and serum IgG antibodies against L.intracellularis. Two in-feed antimicrobial strategies were analysed in this thesis for their ability to induce the development of immunity to L.intracellularis, while avoiding clinical signs of disease. The first strategy evaluated the use of low levels of in-feed antimicrobials to allow subclinical infection and the development of immunity. The second strategy evaluated the use of high levels of in-feed antimicrobials to terminate infection two weeks after exposure to L.intracellularis. Gaining a greater understanding of how L.intracellularis infection is spread both within and between piggeries will enable the development of management strategies to control the spread of infection. This thesis examined the possibility that other species in contact with pigs and piggeries such as rats, mice and birds may transmit infection to pigs. The transmission of infection between pigs via the faecal/oral route was also examined, as was the survival and infectivity of L.intracellularis over time. Ultimately this thesis aimed to understand the pattern of L.intracellularis infection and the survival and transmission of L.intracellularis in order to develop effective control measures for PPE, especially in minimal disease herds.

Zika est un Flavivirus émergent transmis à l’Homme par piqûre de moustique. Il a récemment été à l’origine d’épidémies d’envergure mondiale et représente une menace pour la santé publique. L’infection Zika est souvent asymptomatique ou engendre un syndrome grippal bénin. Cependant, des complications sévères ont été associées au virus Zika, telles qu’un syndrome de Guillain-Barré ou des encéphalites chez l’adulte, ainsi que des malformations congénitales comme la microcéphalie. De nombreux facteurs sont susceptibles d’influencer la sensibilité d’un individu au virus Zika, y compris les variants génétiques de l’hôte.Nous avons étudié le rôle des facteurs génétiques de l’hôte dans sa sensibilité à l’infection par le virus Zika. Pour cela, nous avons utilisé des lignées de souris du Collaborative Cross (CC), une population génétique de référence caractérisée par une diversité génétique aussi vaste que celle des populations humaines.Nous avons d’abord montré que le fond génétique de souris déficientes pour le gène du récepteur à l’interféron de type I (Ifnar1) joue un rôle drastique dans leur sensibilité au virus Zika. La diversité génétique des souris CC, préalablement traitées par un anticorps bloquant le récepteur IFNAR, s’exprime par des phénotypes allant d’une résistance complète jusqu’à des formes sévères de la maladie. L’influence des facteurs génétiques de l’hôte s’exerce sur de nombreux paramètres tels que la virémie, la charge virale et les lésions pathologiques dans le cerveau, et enfin le taux de réplication dans les cellules infectées. Les différences de sensibilité entre lignées CC s’avèrent corrélées entre les Flavivirus Zika, Dengue et West-Nile. Nos analyses génétiques ont montré que de multiples gènes à effets faibles sous-tendent ces variations phénotypiques, reflétant la complexité de la sensibilité au virus Zika dans les populations humaines, et permettent d’exclure un rôle majeur du facteur de résistance Oas1b.Nous avons ensuite cherché des gènes agissant comme modificateurs de la sensibilité chez des souris déficientes pour le gène Ifnar1 dans un croisement F2 entre des souris C57BL/6J et 129S2/SvPas portant la mutation. L’analyse génétique a permis l’identification de deux QTLs (Quantitative Trait Locus), l’un contrôlant le pic de virémie et l’autre la survie. Une étude bio-informatique nous a permis d’identifier quelques gènes candidats.Nous avons également étudié comment les facteurs génétiques de l’hôte impactent la réplication virale dans des fibroblastes embryonnaires murins (MEFs) dérivés d’une série de lignées de souris présentant des phénotypes contrastés en réponse à l’infection Zika. Nous avons identifié une augmentation de la réplication virale tardive dans les MEFs de la lignée CC071, résultant d’un retard à l’activation de la réponse interféron (IFN). Des analyses génétique et transcriptomique ont exclus des déficiences causées par des gènes uniques et ont favorisé l’hypothèse d’une combinatoire de gènes exerçant des effets faibles dans la voie d’induction de la réponse IFN.Pour finir, nous avons caractérisé la réponse IFN induite par le virus Zika dans des neurones primaires murins. Cette étude a montré que la capacité des neurones primaires à limiter la réplication virale est moindre que celle des MEFs en raison d’un retard à l’induction de la réponse IFN. Enfin, les facteurs génétiques de l’hôte exercent un rôle critique dans ce contexte puisque les neurones primaires de CC071 présentent un phénotype extrême par comparaison avec des lignées plus résistantes.Notre travail a mis en évidence le rôle des facteurs génétiques de l’hôte dans la pathogénie de l’infection Zika et illustre le potentiel des souris CC dans des études génétiques aussi bien qu’en tant que nouveaux modèles d’infection. Une analyse poussée des lignées aux phénotypes extrêmes permettra d’élucider les mécanismes génétiques de la sensibilité au virus Zika et améliorera notre compréhension de la maladie chez l’Homme
Zika virus (ZIKV) is a mosquito-transmitted flavivirus responsible for worldwide epidemics and constitutes a major public health threat. The majority of ZIKV infections in humans are either asymptomatic or result in a mild febrile illness. However, some patients develop a more severe, sometimes life-threatening, form of the disease. Recent evidence showed that ZIKV infection can trigger Guillain-Barré syndrome and encephalitis in adults, as well as congenital malformations such as microcephaly. The severity of ZIKV disease in humans depends on many factors, likely including host genetic determinants.We investigated how genome-wide variants could impact the susceptibility to ZIKV infection in mice. To this end, we used mouse strains of the Collaborative Cross (CC), a new genetic reference population encompassing a genetic diversity as broad as that of human populations.First, we described that the susceptibility of Ifnar1 (receptor to type I interferon) knockout mice is largely influenced by their genetic background. We then showed that the genetic diversity of CC mice, which IFNAR was blocked by anti-IFNAR antibody, expressed phenotypes ranging from complete resistance to severe symptoms and death with large variations in the peak and rate of decrease of plasma viral load, in brain viral load, in brain histopathology and in viral replication rate in infected cells. Differences of susceptibility between CC strains were correlated between Zika, Dengue and West Nile viruses. We identified highly susceptible and resistant CC strains as new models to investigate the mechanisms of human ZIKV disease and other flavivirus infections. Genetic analyses revealed that phenotypic variations were driven by multiple genes with small effects, reflecting the complexity of ZIKV disease susceptibility in human population. Notably, our results also ruled out a role of the Oas1b gene in the susceptibility to ZIKV.In a second part, we searched for genes which modify the susceptibility of Ifnar1 knockout mice in an F2 cross between C57BL/6J and 129S2/SvPas mice harboring the mutation. Genetic analysis revealed two Quantitative Trait Locus (QTL) controlling either the peak viremia or the mouse survival. Although these QTLs critical intervals contained hundreds of genes, data mining led us to identify a few candidate causal genes.Then, we investigated how host genetic factors influence viral replication in infected cells using Mouse Embryonic Fibroblasts (MEFs) derived from a series of CC strains with contrasted phenotypes observed in response to ZIKV infection in vivo. MEFs from CC071 strain displayed unique features of increased viral replication rate in late infection. Using transcriptomic analysis, we demonstrated that the phenotype of CC071 infected MEFs resulted from a delayed induction of the type I interferon (IFN) response. Genetic analyses ruled out single gene deficiencies but rather suggested combined effects of multiple factors in the type I IFN induction signaling pathway.Finally, we characterized the ZIKV-induced type I IFN response in MEFs and primary neurons derived from C57BL/6J mouse strain. Primary neurons were less capable than MEFs to control the viral replication due to a delayed IFN response. We later showed that host genetic factors also play a critical role in this context as ZIKV-infected CC071 primary neurons displayed an extreme phenotype compared to neurons from strains that are more resistant.Altogether, our work has unraveled the role of host genes in the pathogeny of ZIKV infection and illustrates the potential of CC mouse strains for genetic studies and as new models of infectious diseases. Extensive analysis of CC strains with extreme phenotypes help us elucidate how genetic variants affect susceptibility as well as immune responses to flaviviral infection and will provide deeper understanding of the pathophysiology of human ZIKV disease

Les légumineuses en milieu carencé en azote, établissent une relation symbiotique avec les bactéries du sol appelées rhizobia. Cette interaction conduit à la formation d’un nouvel organe racinaire, la nodosité. Au sein de celle-ci, les rhizobia se différencient en bactéroïdes fixant l’azote atmosphérique au profit de la plante. La colonisation massive et chronique des cellules symbiotiques de nodosités par les rhizobia ne déclenche aucune réaction de défense visible. Au laboratoire nous avons isolé deux mutants symbiotiques développant des réactions de défense dans les nodosités de Medicago truncatula indiquant qu’il existe un contrôle strict de l’immunité dans cet organe. L’objectif de cette thèse est de comprendre comment l’immunité symbiotique contrôle les voies de signalisation hormonales de défense afin d’héberger le partenaire symbiotique et de trouver de nouveaux outils pour mieux comprendre les mécanismes de défense. Pour cela, des approches moléculaires, pharmacologiques et génétiques sont utilisées. Les résultats obtenus dans cette thèse suggèrent que les mécanismes de défense adoptés par M. truncatula varient en fonction de l’écotype de la plante. L’écotype A17 exploite deux voies de résistance : la voie de la sénescence et la voie des réactions de défense. Cependant, l’écotype R108 n’exploite que la voie des réactions de défense. Ce travail suggère également que chez M. truncatula A17, la protéine SymCRK réprime la voie de signalisation de l’acide jasmonique qui conduit au déclenchement de la sénescence, tandis que la protéine DNF2 réprime principalement la voie de signalisation de l’acide salicylique qui conduit au déclenchement des réactions de défense et aussi réprime secondairement la voie acide jasmonique. Chez M. truncatula R108, les deux protéines SymCRK et DNF2 contrôlent les voies de signalisation de l’acide salicylique et de l’éthylène, avec DNF2 contrôlant préférentiellement la voie acide salicylique et SymCRK contrôlant préférentiellement la voie éthylène. Cette thèse suggère aussi l’implication des protéines R dans le contrôle de l’accommodation intracellulaire des rhizobia. Un gène CNL-5 semble être impliqué dans le contrôle de l’infection des cellules symbiotiques, et deux autres gènes CNL-4 et TNL-2 semblent être impliqués dans le contrôle de l’efficacité de l’infection. Finalement, cette thèse a permis d’isoler une souche bactérienne E. adhaerens, capable se comporter comme un symbiote ou comme un pathogène pour plusieurs espèces de Medicago
When grown under limited nitrogen resources, legumes establish a symbiotic relationship with soil bacteria called rhizobia. This interaction leads to the formation of a new root organ, the nodules. Within nodules, rhizobia differentiate into bacteroids and fix atmospheric nitrogen for the plant. The massive and chronic colonization of nodule symbiotic cells by the rhizobia does not trigger any visible defense reactions. In the laboratory, we previously isolated two symbiotic mutants developing defense reactions in Medicago truncatula nodules, indicating a strict control of the nodule immunity. The thesis project aimed first to understand the relationship between symbiotic genes and immunity mediated by defense hormones and second to find new tools to help to better understand nodules defense mechanisms. For this, molecular, pharmacological and genetic approaches were used. The results obtained in this thesis suggest that the defense mechanisms adopted by M. truncatula vary depending on the plant ecotypes. The A17 ecotype uses two resistance pathways; senescence and defense reactions. While, the R108 ecotype uses defense reactions pathway to fight against rhizobia. This work also suggests that in M. truncatula A17, the SymCRK protein represses jasmonic acid signalling. This work also suggests that in M. truncatula A17, the SymCRK protein represses the jasmonic acid signaling pathway involved in senescence triggering. In contrast, the DNF2 protein mainly represses the salicylic acid signaling pathway involved in defense reactions and also represses the JA pathway. In M. truncatula R108, the two proteins SymCRK and DNF2 control salicylic acid and ethylene signaling pathways involved in triggering defense reactions. DNF2 controls the salicylic acid pathway more than the ethylene signaling pathway, while, SymCRK controls more the ethylene pathway than the salicylic acid signaling pathway in nodules. This thesis also suggests the involvement of R proteins in the control of intracellular accommodation of rhizobia. The intracellular receptor CNL-5 appears to be involved in controlling the infection of symbiotic cells and the receptors CNL-4 and TNL-2 genes appear to be involved in controlling the infection efficacy of the symbiotic cells. Finally, this thesis allowed the identification of a new bacterial strain E. adhaerens, capable of behaving as a symbiont or as a pathogen for several Medicago species. This can be used as a tool to study the nodules defense induction and to understand the rhizobia intracellular accommodation in symbiotic cells

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.

Anopheles gambiae est le vecteur le plus redoutable de Plasmodium falciparum, l'agent principal responsable du paludisme en Afrique Sub-saharienne. Les nouvelles stratégies de lutte contre la maladie visent à limiter ou interrompre le développement du parasite au cours de son cycle de vie chez le moustique vecteur, ce qui nécessite une bonne connaissance des interactions vecteur*parasite. L'objectif principal de cette thèse a été d'évaluer l'impact de facteurs environnementaux et génétiques sur le développement de P. falciparum chez An. gambiae en conditions naturelles de transmission. Pour la réalisation de ce projet, nous avons utilisé un système d'infections expérimentales, où des populations sauvages d'anophèles provenant de différentes localités ont été infectées avec des isolats naturels de P. falciparum. Notre étude a révélé que les moustiques provenant des zones urbaines étaient plus infectés que ceux des zones péri-urbaines, démontrant que la compétence vectorielle est dépendante des interactions vecteur*parasite*environnement. Nous avons ensuite mesuré l'impact de l'environnement aquatique sur la capacité des moustiques adultes à transmettre le parasite en corrélant la composition de la flore bactérienne intestinale des moustiques femelles avec leur statut d'infection à P. falciparum. Nous avons mis en évidence que la flore bactérienne intestinale de moustique différait en fonction du gîte aquatique et qu'une communauté bactérienne, les Enterobacteriaceae, jouait un rôle dans la susceptibilité du moustique à l'infection. Enfin, le polymorphisme génétique de deux gènes de l'immunité ayant un rôle démontré dans l'infection par Plasmodium, TEP1 et APL1A, a été étudié chez nos moustiques sauvages. Nous avons montré que les différents allèles étaient répartis différemment au sein des différentes populations de vecteurs et qu'ils étaient soumis à des forces évolutives. Le rôle des interactions génome*environnement et leurs implications dans la compétence vectorielle seront discutées. En conclusion, les résultats de ce travail de thèse soulignent la complexité des interactions vecteur*parasite qui sous-tendent la compétence vectorielle et montrent l'importance de prendre en compte les facteurs environnementaux pour l'élaboration de nouvelles stratégies de lutte
Anopheles gambiae is the most tremendous vector of Plasmodium falciparum, the major agent of malaria in sub-Saharan Africa. New malaria control approaches envision interrupting transmission cycle in the mosquito, however this will require a better knowledge of vector*parasite interactions. The main objective of this PhD work was to investigate the impact of the environmental and genetic factors on the development of P. falciparum into An. gambiae in natural settings. To carry out this project, we used experimental infection system; wild anopheline mosquito populations from different localities were infected with natural isolates of P. falciparum. Our study revealed that mosquitoes from urban area were more infected than those from sub-urban areas, demonstrating that vector competence depends on vector*parasite*environment interactions. We then measured the impact of the aquatic environment on the adult mosquito capacity to transmit parasites. Correlation analysis between the mosquito gut microbiota and P. falciparum infection status was performed. We showed that mosquito bacterial flora differed according to the aquatic breeding site and that Enterobacteriaceae community was involved in the mosquito susceptibility. Genetic polymorphisms of two immune genes involved in parasitic defense, TEP1 and APL1A, were then studied. We showed that the different alleles were differentially spread into wild vector populations and evolutive forces were acting. Genome*environment interactions and their involvement in vector competence will be discussed. Finally, this thesis highlights the complexity of vector*parasite interactions underlying vectorial competence and pinpoints the importance to take into account environmental factors to elaborate new malaria control strategies

Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.11.13 – Прилади і методи контролю та визначення складу речовин. – Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського», МОН України, Київ, 2019. У роботі здійснено математичне моделювання, яке дозволило визначити раціональні параметри та режими роботи сенсору для забезпечення максимальної чутливості схеми. Досліджено параметри процесу контролю та межі застосування методу. Здійснено моделювання контролю дефектів різних типів. Здійснено моделювання розподілу чутливості ємнісного сенсора, що дозволило порівняти сенсори різної геометричної форми та визначити оптимальні конструктивні параметри. Запропоновано конструкцію сенсора, яка має найвищу чутливість та глибину проникнення. Здійснено моделювання процесу контролю матеріалів зі змінними провідними властивостями, що підтвердило можливість застосування методу для такого класу матеріалів. Розроблено спосіб підвищення завадостійкості та швидкодії методу. Проведено експериментальні дослідження, які підтвердили працездатність електроємнісного методу для контролю дефектів. Шляхом математичного моделювання проаналізовано вплив різних чинників на випадкову похибку вимірювання.

Despite the introduction of antiretroviral therapy, the HIV/HIV epidemic remains an unsolved global health problem. Amongst all the host defence mechanisms, HLA class I molecules have shown the strongest genetic association with delayed disease progression, in particular HLA-B alleles. Numerous studies have shown that the HLAmediated CD8+ T cell responses play a central role in the immune control of HIV. Yet our understanding of HLA-mediated immune control of HIV remains incomplete, even when considering the best-defined epitopes restricted by the protective HLA alleles at a population level. The studies I have conducted and describe herein focus on two well-charaterised protective HLA-B molecules, HLA-B*81:01 and HLA-B*27:05; a third protective molecule, HLA-B*52:01, that has not been well-studied hitherto; and finally the most prevalent HLAB allele in many Asian populations such as Taiwan, HLA-B*40:01, which has an apparently neutral effect on viral replication. This thesis is centred on the Gag-specific immune response, since previous studies have shown the benefits of CD8+ T-cell responses targeting this conserved and immunogenic region of the HIV proteome, in particular the p24 capsid protein. I have investigated here HLA footprints driven by CD8+ T-cell pressure on HIV that are evident in the viral sequences of individuals expressing these HLA molecules. These footprints include novel escape and putative compensatory mutations. The impact of these variants on viral replicative capacity (VRC) and on HIV disease outcome clinical outcomes was examined via fitness assays. These studies identified several escape mutations that effectively cripple HIV. The distinct compensatory pathways available to the virus to mitigate the fitness cost of particular escape mutations were evaluated. In the course of these analyses I have demonstrated the critical influence of the viral backbone, including HIV clade, in combination with particular viral variants, on VRC. Computational modelling analysis has been applied to facilitate understanding of the mechanism by which certain mutants affect the stability of interactions between HLA and viral capsid protein. This thesis offers novel insights into immune control of the key HIV subtypes – B- and C-clade – and of the most severely affected populations – in Africa (South Africa) and Asia (India and Taiwan) – within the global epidemic. This work helps to better define the viral mutation landscape that is essential both for future vaccines designed to corner the virus, and for successful HIV cure strategies.

Les arbovirus (virus transmis par des arthropodes) sont à l'origine de maladies humaines telles que la dengue, le chikungunya ou encore le Zika. Le moustique Aedes aegypti, est le vecteur majeur de ces trois arbovirus. La faible efficacité des méthodes de contrôle des populations de moustiques, principalement réalisées au moyen d'insecticides chimiques ouvre un champ de développement de nouvelles approches en lutte antivectorielle. Le moustique, hôte vecteur, contrôle la réplication virale en limitant les réponses immunitaires antivirales. La machinerie RNA interférence (RNAi) est la voie jouant un rôle majeur dans l'immunité antivirale chez le moustique. Alors que le rôle des deux voies, siRNA (" small interfering RNA ") et piRNA (" piwi-interfering RNA "), est de mieux en mieux compris dans les réactions antivirales du vecteur, peu de connaissances sont disponibles à ce jour en ce qui concernent les interactions entre la voie miRNA (" micro RNA ") et les arbovirus. Ainsi, nous proposons une analyse détaillée des mécanismes par lesquels les miARN tentent de réguler la réplication virale chez le moustique. Dans la première partie de la thèse, nous avons effectué une analyse génomique pour identifier les miRNAs pouvant interagir chez Ae. aegypti avec divers lignées/génotypes des virus chikungunya (CHIKV), de dengue (DENV) et de Zika. Avec l'aide d'outils de prédiction faisant appel à divers algorithmes, plusieurs sites de liaison de miARN avec différents lignées/génotypes de chaque arbovirus ont été identifiés. Nous avons ensuite sélectionné les miARN pouvant cibler plus d'un arbovirus et nécessitant un faible seuil d'énergie lors de la formation des complexes entre l'ARNm
Mosquito-borne arboviruses cause some of the world’s most devastating diseases and are responsible for recent dengue, chikungunya and Zika pandemics. The yellow-fever mosquito. Aedes aegypti, plays an important role in the transmission of all three viruses. The ineffectiveness of chemical control methods targeting Ae. aegypti makes urgent the need for novel vector-based approaches for controlling these diseases. Mosquitoes control arbovirus replication by triggering immune responses. RNAi machinery is the most significant pathway playing a role on antiviral immunity. Although the role of exogenous siRNA and piRNA pathways in mosquito antiviral immunity is increasingly better understood, there is still little knowledge regarding interactions between the mosquito cellular miRNA pathway and arboviruses. Thus further analysis of mechanisms by which miRNAs may regulate arbovirus replication in mosquitoes is pivotal. In the first part of the thesis, we carried out genomic analysis to identify Ae. aegypti miRNAs that potentially interact with various lineages and genotypes of chikungunya (CHIKV), dengue (DENV) and Zika viruses. By using prediction tools with distinct algorithms, several miRNA binding sites were commonly found within different genotypes/and or lineages of each arbovirus. We further analyzed the miRNAs that could target more than one arbovirus and required a low energy threshold to form miRNA-vRNA (viral RNA) complexes and predicted potential RNA structures using RNAhybrid software. Thus, we predicted miRNA candidates that might participate in regulating arboviral replication in Ae. aegypti. In the second part of the thesis, we developed a miRNA-based approach that results in a dual resistance phenotype in mosquitoes to dengue serotype 3 (DENV-3) and chikungunya (CHIKV) viruses for stopping arboviruses spreading within urban cycles. The target viruses are from two distinct arboviral families and the antiviral mechanism is designed to function through the endogenous miRNA pathway in infected mosquitoes. Ten artificial antiviral 4 miRNAs capable of targeting ~97% of all published strains were designed based on derived consensus sequences of CHIKV and DENV-3. The antiviral miRNA constructs were placed under control of either an Aedes PolyUbiquitin (PUb) or Carboxypeptidase A (AeCPA) gene promoter triggering respectively expression ubiquitously in the transgenic mosquitoes or more locally in the midgut epithelial cells following a blood meal. Challenge experiments using viruses added in blood meals showed subsequent reductions in viral transmission efficiency in the saliva of transgenic mosquitoes as a result of lowered infection rate and dissemination efficiency. Several components of mosquito fitness, including larval development time, larval/pupal mortality, adult lifespan, sex ratio, and male mating competitiveness, were examined: transgenic mosquitoes with the PUb promoter showed minor fitness costs at all developing stages whereas those based on AeCPA exhibited a high fitness cost. Further development of these strains with gene editing tools could make them candidates for releases in population replacement strategies for sustainable control of multiple arbovirus diseases

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The immune system matures progressively from infancy to adulthood, thus children may differ from adults in their immune function. The immature immune system demonstrates a higher naive to memory T cell ratio, defective macrophage function and antigen presentation which, cumulatively, results in diminished production of cytokines such as IFN-y. This cytokine has been shown to play a pivotal role in protection against Mycobacterium tuberculosis (M. tuberculosis) disease. Other cytokines, such as IL-12 and TNF-a, are also involved in the defence against M. tuberculosis. Epidemiological evidence suggests an agerelated incidence of tuberculosis (TB) irrespective of prevalence in a given region. Reports in the literature also demonstrate depressed immune responses in TB patients, at diagnosis, (before TB therapy) with subsequent improvement after TB therapy. The aims of this study were to optimise a whole blood assay in order to characterise immune responses, as measured by proliferation and cytokine production, in TB patients (after TB therapy) and healthy controls of different ages. Immune responses of TB patients would also be compared, before, and after TB therapy. A total of 68 subjects were included in this study. These comprised 27 TB patients and 41 healthy Mantoux positive controls. All subjects were stratified into two age groups: <12 years and >12 years. Diluted whole blood was cultured and stimulated with the mitogen, phytohaemagglutinin (PHA) and the specific mycobacterial antigen, purified protein derivative (PPD) to measure proliferation and IFN-y, IL-2, TNF-a and IL-10 production in the supernatant of cultures. Age was a significant variable for the following PHA-stimulated cytokines: IFN-y, TNF-a and IL-10. Proliferation and IL-2 production after PHA stimulation did not demonstrate any relationship with age. None of the PPD-stimulated proliferative or cytokine responses demonstrated any correlation with age. Concentrations of PHA- and PPD-induced IFN-y for all subjects (patients and controls) were increased “after therapy”, compared to “before therapy”. This phenomenon could possibly be due to maturation in the capacity of the immune system to produce this cytokine. Patients >12yrs demonstrated improvement in all proliferative and cytokine responses (except for PPD-induced IL-2 and TNF-a) “after therapy”, compared to “before therapy”. This is probably a valid finding and is thus in accordance with the literature. The whole blood assay is a simple, non-laborious assay that, according to the literature, produces results that seem to correlate well with that of conventionally used PBMCs. Age appears to be an important variable in the quantitative assessment of cellular immune responses (when the mitogen, PHA is used as a stimulant) and immune responses of older TB patients appear to improve after TB therapy, compared to before TB therapy.
AFRIKAANSE OPSOMMING: Die immuunsisteem matureer stelselmatig van kind na volwassene. Dus sal kinders se immuniteit verskil van volwassenes s’n. Die immature immuunsisteem het ‘n hoer nai'witeit vir geheue T-sel verhouding, defektiewe makrofaag funksie en antigeen presentering wat gesamentlik lei tot verminderde produksie van sitokiene soos byvoorbeeld IFN-y. Daar is bewys dat hierdie sitokien ‘n deurslaggewende rol speel in die beskerming teen Mycobacterium tuberculosis (M. tuberculosis). Ander sitokiene, soos IL-12 en TNF-a speel ook ‘n rol in die beskerming teen M. tuberculosis. Epidemiologiese data dui aan dat daar ‘n ouderdomverwante insidensie van tuberkulose (TB) is sonder dat dit beinvloed word deur die voorkoms van TB in ‘n sekere area. Verslae in die literatuur wys ook op onderdrukte immuniteitrespons in TB-pasiente by diagnose (voor TB-behandeling) met uiteindelike verbetering na TB-behandeling. Die doel van hierdie studie was om ’n volbloed metode te optimaliseer in ’n poging om die immuunrespons te karakteriseer soos gemeet met behulp van proliferasie en sitokien produksie by TB-pasiente (na TB-behandeling) en gesonde kontrole persone van verskillende ouderdomme. Die immuunrespons van TB-pasiente word ook vergelyk voor en na TBbehandeling. ‘n Totaal van 68 gevalle is vir die studie gebruik. Dit sluit in 27 TB-pasiente en 41 gesonde Mantoux positiewe kontroles. A1 die gevalle is in twee ouderdomsgroepe verdeel: <12 jaar en >12 jaar. Kulture is gemaak van verdunde volbloed en gestimuleer met phytohaemaglutinin (PHA) en gesuiwerde proteien derivaat (purified protein derivative-PPD) om proliferasie en IFN-y, IL- 2, TNF-a en IL-10- produksie in die supernatant van die kulture te meet. Ouderdom was ‘n beduidende veranderlike vir die volgende PHA-gestimuleerde sitokiene: IFN-y, TNF- a en IL-10. Daar was geen korrelasie tussen proliferasie en IL-2-produksie na PHA-stimulasie aan die een kant en ouderdom aan die ander kant nie. Geen van die PPDgestimuleerde proliferasie response of sitokien response het enige korrelasie met ouderdom getoon nie. Konsentrasies van PHA- en PPD-geinduseerde IFN-y vir alle gevalle (pasiente en kontrole) was verhoog “na behandeling”, vergeleke met “voor behandeling”. Hierdie fenomeen kan moontlik toegeskryf word aan maturasie in die vermoe van die immuunsisteem om sitokiene te vervaardig. Pasiente >12 jaar het bewyse getoon van verbetering in alle proliferasie en sitokien response (behalwe vir PPD-gei'nduseerde IL-2 en TNF-a) “na behandeling”, vergeleke met “voor behandeling”. Dit is waarskynlik ‘n geldige bevinding en is dus in ooreenstemming met verslae in die literatuur. Die volbloed metode is ‘n eenvoudige metode wat nie baie arbeidsintensief is nie, wat volgens die literatuur, resultate lewer wat goed korreleer met die konvensionele gebruik van perifere bloed mononukliere selle (PBMC’s). Dit wil voorkom asof ouderdom ‘n belangrike veranderlike is in die kwantitatiewe beoordeling van sellulere immuunrespons (wanneer PHA gebruik word as ‘n stimulant), en of die immuunrespons van ouer TB-pasiente verbeter na TB-behandeling in vergeleke met die respons voor TB-behandeling.

[ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales.
[CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals.
[EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections.
The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM).
Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035
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