This research was performed to determine; (1) the prevalence of Toxoplasma antibodies among starlings in Northern California, (2) if such starlings can transmit the disease, and (3) if tissues from seropositive starlings contain encysted forms of T. gondii.

Phylum Apicomplexa comprises several thousand parasitic protozoans that cause significant disease in humans and animals worldwide. Of particular relevance to human health are Plasmodium spp., the causative agents of malaria; and Toxoplasma gondii, which infects approximately 30% of all humans on earth, and causes serious disease in immunocompromised individuals and neonatally infected fetuses. Central to the pathogenesis of apicomplexans is a unique form of substrate-dependent locomotion termed “gliding motility”, which is essential for traversing the environment and actively invading host cells. Driving motility is the class-XIV unconventional myosin motor (MyoA), which is notably divergent from canonical myosins in that it lacks a “tail” and conventional sequence motifs in both the neck and motor regions. Thus, the mechanisms that enable MyoA to function with a step size and velocity similar to canonical human myosins are not well understood. Over the past 2 decades, the apicomplexan research community has identified many of the components involved in gliding motility, resulting in a functional model of MyoA and accessory proteins forming the “glideosome” macromolecular complex. However, there was still relatively little known about the unique physical processes that drive force production and transduction in the apicomplexan motor complex. Thus, I set out to use structural and biophysical methods to interrogate this divergent molecular motor, and provide the first high-resolution model of apicomplexan motility. Towards this goal, I first used structural and biophysical methods to establish the most complete model to date of class-XIV motor complex assembly, answering key questions about the interface between MyoA and its accessory proteins. To understand the unique molecular basis of force production in apicomplexan motors, I then solved the first ever crystal structure of a class-XIV myosin, MyoA from T. gondii. Supplementing this structure with further biophysical data, I was able to determine the functional consequences of class-defining sequence polymorphisms, and elucidate the basis of phosphorylation-dependent motor regulation. The systematic dissection of apicomplexan motor complexes described herein provides crucial insight into a fundamental biological process, and may help overcome existing barriers for targeted therapeutic development. / Graduate

Estima la frecuencia de anticuerpos contra toxoplasma gondii y Neospora caninum en vicuñas de la provincia de Yauli–Junín, así como también determinar el grado de concordancia entre las pruebas de inmunofluorescencia indirecta (IFI) y ELISA indirecta. Se colectaron 79 muestras de sangre de vicuñas, obtenidas durante un “Chaku”, técnica ancestral de arreo que permite la captura de animales vivos para la esquila, realizado en el distrito de Paccha provincia de Yauli, ubicado en el departamento de Junín. Las muestras fueron trasladadas al laboratorio de Microbiología y Parasitología Sección Parasitología de la Facultad de Medicina Veterinaria de la UNMSM, para su posterior análisis. El diagnóstico para ambas enfermedades se realizó utilizando las pruebas de ELISA indirecta e IFI, en cada caso. La frecuencia hallada para Toxoplasma gondii fue de 5.06 y 3.08% por las pruebas de ELISA indirecta y la de IFI respectivamente; no encontrándose reacción seropositiva para Neospora caninum con las dos pruebas usadas. Con relación al grado de asociación, entre las dos pruebas usadas en el diagnóstico de Toxoplasma gondii se encontró un valor de kappa de 0.85, lo cual significa que la concordancia ofrecida por ambas pruebas es muy buena. / Tesis

Introduction: The intracellular protozoan Toxoplasma gondii is a uniquely successful parasite that globally exploits a wide host range encompassing essentially all warm-blooded animals including both mammalian and avian species. Felidae are the only definitive hosts in the facultative heteroxenous life cycle. Pathogenesis of T. gondii infection has been studied mainly in mammals. There is only rare information on the immune reaction in chickens although birds are known to be important hosts. Considering that the host’s peripheral blood cells serve as target and thus interact with the intracellular parasite during distribution in the infected host, invasion and intracellular replication of the tachyzoites of T. gondii in different mononuclear cell populations isolated from chicken peripheral blood (erythrocytes, thrombocytes, monocyte-derived macrophages (MM)) were studied. Objectives: Peripheral blood mononuclear cells (PBMCs), namely thrombocytes and erythrocytes as well as MM were characterized concerning their capability to host T. gondii. Primary cell infection models were established in order to assess parasite replication. In MM two different strains, ME49 (strain type II) and NED (strain type III), were compared for their replication rate. Cellular response to infection with ME49 was studied in primary MM. Furthermore, the response of the primary macrophages to infection with the ME49 strain was observed by evaluation of gene expression of IL-1ß, IL-12p40, Lipopolysaccharide induced TNF-α factor (LITAF) and inducible nitric oxide synthase (iNOS). Gene transcription data were normalized to those of GAPDH and G6PDH. Parasite replication as well as the fold change of cytokines were statistically evaluated. Results: Primary cultures containing at least 80 % of the respective target cells and suitable for long-term observations were produced by the procedures established in the current study. It was demonstrated that cultivation at 40°C (representing the physiological body temperature in chickens) and using of primary avian cells instead of mammalian cell lines such as Vero or HD11 as host cells distinctly affected parasite replication. T. gondii tachyzoite numbers significantly increased in MM (300 percent at 48 h p.i., ME49) during long-term investigation over 72 h but with initial and final decrease. In comparison, the number of parasite stages increased 1000 percent in Vero and 300 percent in HD11 by only robust increases (ME49), respectively. In contrast, the number of parasite stages significantly decreased in primary erythrocytes and thrombocytes down to less than 20 percent not increasing again. No significant differences were detected between strain type II (ME49) and III (NED) regarding their replication potential in MM. The mRNA expression for cytokines and iNOS of MM infected with viable ME49 tachyzoites was significantly different from uninfected MM and MM incubated with heat-inactivated ME49 tachyzoites. Especially at 8 to 24 h p.i., parasite replication coincided with upregulation of gene expression (IL-1ß, LITAF, iNOS) in infected MM. For infection with heat-inactivated tachyzoites, mRNA regulation increased directly after infection with subsequent decrease between 4 to 12 h p.i. and in this entirely, level of fold change for IL-1 ß was significantly higher (5 times as high), but lower for iNOS (approximately half). In comparison, mRNA upregulation of IL-12p40 was delayed for both with only one peak for infection with heat-inactivated tachyzoites at 8 h p.i., but repeated increase (8, 24, 36 h p.i.) for viable ME49 being in total almost twice as high (at 36 h p.i.). Conclusion: Primary chicken MM are a suitable host cell system to study immune reaction to T. gondii tachyzoite infection, while erythrocytes and thrombocytes are not supporting parasite replication. Avian MM are likely an important vehicle for distributing the parasite from blood vessels into different organs and thus promote infection. In addition to this putative Trojan horse function, MM react to pathogen invasion by pro-inflammatory upregulation of IL-1ß, IL-12p40, LITAF and iNOS. Activation of these mediators points to contribution of MM to immunity against T. gondii in chicken. These seemingly contradictory findings during T. gondii infection of MM needs further investigation. It was hypothesized that nucleated avian erythrocytes and thrombocytes might also serve as host cells, however, no parasite replication could be demonstrated in these cells. Nevertheless, they may though play a role in the progress and systemic distribution of infection which should be investigated in more detail.:1 INTRODUCTION ............................................................................... 1 1.1 TOXOPLASMA GONDII................................................................................... 1 1.1.1 Structures and life cycle .............................................................................. 1 1.1.2 Life cycle .................................................................................................... 3 1.1.3 Genotypes.................................................................................................. 4 1.2 ZOONOSIS .................................................................................................... 5 1.3 RELEVANT DIFFERENCES OF AVIAN SPECIES FROM MAMMALS AS IH ..... 7 1.4 T. GONDII INFECTION IN CHICKENS ............................................................. 8 1.5 AIMS AND FOCUSES OF THIS STUDY .......................................................... 9 2 RESULTS........................................................................................ 10 2.1 1 ST PUBLICATION ...................................................................................... 10 2.2 2ND PUBLICATION ....................................................................................... 19 2.3 3RD PUBLICATION: ...................................................................................... 29 3 DISCUSSION .................................................................................. 38 3.1 ESTABLISHMENT OF PRIMARY BLOOD CELL CULTURES ......................... 38 3.2 CHICKEN ERYTHROCYTES AND THROMBOCYTES.................................... 40 3.3 MONOCYTE-DERIVED MACROPHAGES (MM) ............................................ 41 3.4 T. GONDII STRAIN DIVERSITY .................................................. .................. 44 3.5 PRIMARY CELLS AS A TOOL FOR T. GONDII RESEARCH ......................... 46 3.6 OUTLOOK..................................... ............................................................... 47 3.7 CONCLUSIONS ............................................................................................ 48 4 SUMMARY........................................................................ .............. 49 5 ZUSAMMENFASSUNG ........................................................ .......... 51 6 REFERENCE LIST................................................................ .......... 53 / Einleitung: Der intrazellulär-lebende Protozoe Toxoplasma gondii ist einzigartig erfolgreich. Er nutzt weltweit eine große Bandbreite verschiedener Wirtsspezies, zu denen im Wesentlichen alle warmblütigen Tiere, vor allem Säugetiere und Vögel, zählen. Feliden sind die alleinigen Endwirte im fakultativ heteroxenen Lebenszyklus. Die Pathogenese der T. gondii-Infektion wurde hauptsächlich im Säugetier betrachtet. Über die Immunantwort im Huhn gibt es nur wenige Informationen, obwohl Vögel als Wirtsspezies eine große Bedeutung haben. Wegen der Schlüsselrolle, welche den peripheren Blutzellen des Wirtes im Verlauf der Infektion hinsichtlich der Verteilung des eindringenden Parasiten im Wirt zukommt, wurden Invasion und Vermehrung von T. gondii-Tachyzoiten in verschiedenen mononukleären Zellpopulationen (Erythrozyten, Thrombozyten, Monozyten-abstammende Makrophagen (MM)) betrachtet. Ziele der Arbeit: Periphere mononukleärer Blutzellen (PBMCs), im Detail Erythrozyten, Thrombozyten und MM, sollten hinsichtlich ihrer Empfänglichkeit für T. gondii charakterisiert werden. Zur Prüfung der Parasitenvermehrung wurden Infektionsmodelle mit Primärzellen etabliert. In MM wurden zudem zwei verschiedene Stämme (ME49, Typ II und NED, Typ III) in ihrer Replikationsrate verglichen und die zelluläre Immunantwort (ME49) untersucht. Die Immunantwort der MM wurde mittels Genexpressionsanalyse von IL-1 ß, IL-12p40, Lipopolysaccharid-induzierter TNF-α Faktor (LITAF) und der induzierbaren Stickstoffmonooxid-Synthase (iNOS) untersucht und die Infektion lebender mit Hitze-inaktivierten Tachyzoiten (ME49) verglichen (über 36 h p.i.). Die Transkription der Zielgene wurde gegen GAPDH und G6PDH normalisiert und diese Daten sowie die der Parasitenvermehrung statistisch evaluiert. Ergebnisse: Die hier entwickelten Methoden resultieren in Primärzellkulturen mit ausreichender Reinheit (Anteil Zielzellen ≥ 80 Prozent) und Verwendbarkeit. Die Bedeutung von 40°C (physiologische Körpertemperatur Huhn) als Kultivierungstemperatur sowie der Verwendung von Primärzellen anstatt immortalisierter Zelllinien (Vero, HD11) für die Parasitenvermehrung von T. gondii konnten gezeigt werden. Der signifikante Anstieg der Tachyzoitenzahl in MM über 72 h Betrachtung (etwa 300 Prozent 48h p.i., ME49) war begleitet von initialem und finalem Abfallen. Im Vergleich dazu haben Vero-Kulturen (1000 Prozent Vermehrung, ME49) und HD11 (300 Prozent Vermehrung, ME49) einen linearen Anstieg. Infizierte Erythrozyten und Thrombozyten zeigen im Gegensatz zu MM einen signifikanten Abfall (≤ 20 Prozent bei 12 h p.i.) der Parasitenstadien ohne erneuten Wiederanstieg. In MM wurden für die beiden untersuchten Tachyzoiten-Stämme (ME49, NED) keine signifikanten Unterschiede in der arasitenvermehrung festgestellt. Die Genexpressionsanalyse der Zytokine und iNOS zeigte im Vergleich signifikante Unterschiede zwischen uninfizierten bzw. mit Hitze-inaktivierten Tachyzoiten infizierten MM zu den mit lebenden Stadien infizierten MM. Insbesondere im Zeitraum 8 bis 24 h p.i. überschneiden sich diese Feststellungen zur Hochregulation der Genexpression (IL-1 ß, LITAF, iNOS) mit der detektierten Parasitenvermehrung in lebend-infizierten MM. Hitze-inaktivierte Tachyzoiten steigern die Genexpression direkt nach der Infektion mit anschließendem Abfall (4 bis 12 h p.i.), wobei im Vergleich für IL-1 ß ein signifikant höherer Anstieg (5 facher fold change), aber ein deutlich niedrigerer für iNOS (etwa halber fold change) festgestellt wurde. Die Regulation von IL-12p40 war verspätet mit nur einem Anstieg (8 h p.i.) bei Infektion mit Hiltze-inaktivierten Tachyzoiten, aber wiederholtem Anstieg (8, 24, 36 h p.i.) bei lebenden ME49 (etwa zweifacher fold change). Schlussfolgerungen: Primäre Hühner-MM sind als Wirtszell-System im Infektionsversuch mit T. gondii-Tachyzoiten gut geeignet für Studien zur Immunantwort. Im Gegensatz dazu sind Erythrozyten und Thrombozyten durch fehlende Parasitenvermehrung ungeeignet. Die Empfänglichkeit der MM für Tachyzoiten weist auf Ihre besondere Bedeutung im Verlauf der Verbreitung des Parasiten im Wirtsorganismus hin. Unabhängig von dieser Funktion als Trojanisches Pferd sind MM gleichzeitig von Bedeutung für die Vermittlung der Immunantwort, wie die Hochregulation von IL-1 ß, IL-12p40, LITAF and iNOS als Mediatoren einer proinflammatorischen Aktivierung zeigen. Diese zunächst gegensätzlich erscheinenden Erkenntnisse benötigen weitere Betrachtung. Darüber hinaus sollte die Bedeutung von Erythrozyten und Thrombozyten für den Verlauf der T. gondii-Infektion im Wirt trotz fehlender Wirtszelleignung genauer untersucht werden.:1 INTRODUCTION ............................................................................... 1 1.1 TOXOPLASMA GONDII................................................................................... 1 1.1.1 Structures and life cycle .............................................................................. 1 1.1.2 Life cycle .................................................................................................... 3 1.1.3 Genotypes.................................................................................................. 4 1.2 ZOONOSIS .................................................................................................... 5 1.3 RELEVANT DIFFERENCES OF AVIAN SPECIES FROM MAMMALS AS IH ..... 7 1.4 T. GONDII INFECTION IN CHICKENS ............................................................. 8 1.5 AIMS AND FOCUSES OF THIS STUDY .......................................................... 9 2 RESULTS........................................................................................ 10 2.1 1 ST PUBLICATION ...................................................................................... 10 2.2 2ND PUBLICATION ....................................................................................... 19 2.3 3RD PUBLICATION: ...................................................................................... 29 3 DISCUSSION .................................................................................. 38 3.1 ESTABLISHMENT OF PRIMARY BLOOD CELL CULTURES ......................... 38 3.2 CHICKEN ERYTHROCYTES AND THROMBOCYTES.................................... 40 3.3 MONOCYTE-DERIVED MACROPHAGES (MM) ............................................ 41 3.4 T. GONDII STRAIN DIVERSITY .................................................. .................. 44 3.5 PRIMARY CELLS AS A TOOL FOR T. GONDII RESEARCH ......................... 46 3.6 OUTLOOK..................................... ............................................................... 47 3.7 CONCLUSIONS ............................................................................................ 48 4 SUMMARY........................................................................ .............. 49 5 ZUSAMMENFASSUNG ........................................................ .......... 51 6 REFERENCE LIST................................................................ .......... 53

In-vitro-Koinfektionsstudien mit Toxoplasma gondii und Eimeria tenella in primären Hühnermakrophagen Institut für Parasitologie der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Feburar 2020 91 Seiten, 3 Publikationen, 2 eingreichte Manuskripte, 17 Abbildungen, 5 Tabellen, 188 Literaturangaben Schlüsselwörter: Toxoplasma gondii, Eimeria tenella, Koinfektion, Makrophagen, in vitro, Wirt-Parasit-Interaktion, Parasitenwachstum, Wirtsimmunantwort, Cell-imaging Einleitung: Toxoplasma gondii und Eimeria tenella sind intrazelluläre apikomplexe Parasiten, die in Hühnern weit verbreitet sind. Während Infektionen mit dem zoonotischen Erreger T. gondii beim Geflügel im Allgemeinen subklinisch verlaufen, kann E. tenella in Hühnerbeständen schwere Erkrankungen und wirtschaftliche Verluste verursachen. Aufgrund der hohen Seroprävalenz beider Parasiten bei Hühnern sind Koinfektionen wahrscheinlich. Hühnermakrophagen sind wichtig für die Wirtsimmunantwort gegen diese beiden Protozoen. Es ist jedoch wenig über die Wirt-Parasit- sowie Parasit-Parasit-Interaktion in Hühnermakrophagen während einer Koinfektion bekannt. Ziele der Arbeit: Ein geeignetes In-vitro-Koinfektionsmodell für T. gondii- und E. tenella-Infektionen in primären Hühnermakrophagen wurde erstellt und angewendet, um die Makrophagenantwort auf beide Parasitenarten und Koinfektionen zu untersuchen. Tiere, Material und Methoden: Von Monozyten abgeleitete Makrophagen wurden aus Hühnerblut isoliert und aufgereinigt. Eine Koinfektion mit zwei Tachyzoiten des RH-Stammes von T. gondii und zwei Sporozoiten des E. tenella-Houghton-Stammes pro Zelle wurde gleichzeitig oder nacheinander in vitro durchgeführt. Morphologische Veränderungen der Makrophagen und die Parasitenentwicklung wurden mittels Konfokalmikroskopie (CLSM) 2, 6, 12, 24, 48 und 72 Stunden nach Infektion (hpi) visualisiert. Die Parasitenvermehrung wurde evaluiert, indem die Expression des 529-bp-Fragments von T. gondii und des ITS-1-Genfragments von E. tenella durch qPCR bewertet wurden. Die Makrophagen-Phagozytose wurde durch Exposition gegenüber pH-sensitiven fluoreszierenden Biopartikeln stimuliert und durch ein dreidimensionales Modell unter Verwendung von CLSM und Imaris®-Software 2, 6, 12 und 24 hpi quantifiziert. Weiterhin wurden während der Infektion relevante Zytokine (IL-6, IL-10, IL-12, iNOS, TNF-α und IFN-γ) durch Genexpressionsanalyse 24, 48 und 72 hpi untersucht. Zusätzlich wurden die Zellinvasion durch und das Überleben von T. gondii im Verlauf einer sequentiellen Infektion evaluiert, indem Makrophagen zuvor der Infektion mit E. tenella ausgesetzt waren. Die Motilität invasiver Tachyzoiten wurde innerhalb von 20 hpi durch Live-Cell-Imaging verfolgt. Ergebnisse: Die Makrophagen zeigten eine deutliche immunologische Reaktion und Phagozytoseaktivierung ab 2 hpi. Signifikante Veränderungen der Morphologie mit erhöhter Zellvakuolisierung und -ablösung wurden ab 24 hpi während der Koinfektion beobachtet. Bei zuvor E. tenella-exponierten Makrophagen fiel auf, dass T. gondii bei hoher Motilität über 4 Stunden an der Makrophagenmembran anhaftete, bevor es zu einer Penetration kam. Ab 24 hpi vermehrten sich T. gondii in koinfizierten Makrophagen besser als E. tenella. Eine Koinfektion hemmte die Phagozytoseaktivität von Makrophagen nach 2 hpi erheblich, so dass Biopartikel nicht aufgenommen wurden (12 hpi). Mittels qPCR wurde gezeigt, dass bei sequenzieller Koinfektion 2 hpi circa 4-fach weniger T. gondii überlebten als bei Monoinfektion (P < 0,05). Die Anzahl beider Parasiten nahm während der simultanen Koinfektion zu, aber bei der sequentiellen Koinfektion war die Vermehrung von T. gondii im Vergleich zur Monoinfektion bis 24 hpi auf etwa die Hälfte reduziert. Bis 72 hpi verdoppelte sich die Anzahl von E. tenella, während T. gondii bei Koinfektion in diesem Zeitraum auf dem gleichen Niveau wie 48 hpi blieb. Die mRNA-Expression von iNOS (48 hpi), TNF-α (72 hpi) und von IL-10 (48 hpi und 72 hpi) war während der Koinfektion im Vergleich zur E. tenella Monoinfektion signifikant (P < 0,05) erhöht. Schlussfolgerungen: In dem Koinfektionsmodell wurde eine Interaktion zwischen T. gondii und E. tenella beobachtet. Zusätzlich zu den morphologischen Veränderungen und der Phagozytosehemmung der Makrophagen unterschieden sich die Parasitenvermehrung sowie die Zytokinexpression zwischen Koinfektion und Monoinfektion. E. tenella beeinträchtigt das aktive Eindringen von T. gondii in Wirtszellen, wie dies erfolgt, ist derzeit noch nicht geklärt. / In vitro co-infection studies on Toxoplasma gondii and Eimeria tenella in primary poultry macrophages Institute of Parasitology, Faculty of Veterinary Medicine, University of Leipzig Submitted in February 2020 91 pages, 3 publications, 2 submitted manuscripts, 17 figures, 5 tables, 188 references Keywords: Toxoplasma gondii, Eimeria tenella, co-infection, macrophages, in vitro, host-parasite interaction, parasite growth, host immune response, cell imaging. Introduction: Toxoplasma gondii and Eimeria tenella are intracellular apicomplexan parasites that are widely distributed in chicken. Whereas infections with the zoonotic pathogen T. gondii are generally subclinical in poultry, E. tenella may cause severe disease and economical loss in chicken herds. Due to the high seroprevelences with both parasites in chicken, co-infections are likely to exist. Chicken macrophages are essential in the host innate immune response against these two protozoa. However, little is known about the host-parasite and parasite-parasite interaction in chicken macrophages during co-infection. Objectives: A suitable in vitro model for both T. gondii and E. tenella infection in chicken primary macrophages was established and applied to study the course of infection in mono- or co-infection. Animals, materials and methods: Monocyte-derived macrophages were isolated and purified from chicken blood. Co-infection with two T. gondii RH strain tachyzoites and two E. tenella Houghton strain sporozoites per cell were performed simultaneously or sequentially in vitro. Morphologic alterations of macrophages and parasite development were visualized by confocal laser scanning microscopy (CLSM) at 2, 6, 12, 24, 48 and 72 hours post infection (hpi). Parasite growth was evaluated by assessing expression of the 529-bp fragment of T. gondii and ITS-1 gene fragment of E. tenella by qPCR in parallel. Macrophage phagocytosis was stimulated by exposure to pH sensitive fluorescent bioparticles and quantified by a three-dimensional model using CLSM and Imaris® software at 2, 6, 12 and 24 hpi. Furthermore, infection-related cytokines (IL-6, IL-10, IL-12, iNOS, TNF-α and IFN-γ) were evaluated by gene expression analysis at 24, 48, 72 hpi. The course of sequential infection was evaluated to determine cell invasion and survival of T. gondii in macrophages previously exposed to E. tenella sporozoites. Motility of invasive stages was tracked at the early phase of infection (within 20 hpi) by real time life cell imaging. Results：By cell imaging, macrophages displayed distinctly immunologic activation and phagocytosis at 2 hpi and thereafter. Significant changes of morphology with increased cell vacuolation and detachment were observed on 24 hpi during co-infection. T. gondii tachyzoites adhered for more than 4 hours to host cells displaying high motility instead of cell entry during the early sequential co-infection. However, T. gondii showed better replication than E. tenella in co-infected macrophages from 24 hpi onwards. Co-infection caused inhibition of phagocytosis by macrophages and bioparticles were not incorporated into co-infected cells at 12 hpi by CLSM. By qPCR, it was shown that approximately 4-fold less T. gondii survived in sequentially co-infected cultures at 2 hpi as compared to mono-infection (P < 0.05). Replication of both parasites increased during simultaneous co-infection whereas only half numbers of replicated T. gondii was found in sequential co-infection compared to mono-infection at 24 hpi. At the end of the study period (72 hpi), E. tenella multiplication tended to double increase while T. gondii replication was not distinctly altered during co-infection compared to 48 hpi. Expression of mRNA for iNOS at 48 hpi, for TNF-α at 72 hpi and for IL-10 at 48 and 72 hpi was significantly elevated during co-infection compared to mono-infection (P < 0.05). Conclusions：Mutual interaction between T. gondii and E. tenella were observed in the selected co-infection model. Increased macrophage stress may explain vacuolization and phagocytosis inhibition. In addition to morphologic alterations of macrophages, cytokine up/down- regulation differed between co-infected and mono-infected macrophage cultures. It appears that E. tenella, impairs the active penetration of T. gondii into host cells which deserves further study. The established in vitro infection model may serve to investigate host immune response of macrophages to diverse intracellular pathogens that infect chicken.

Thesis advisor: Marc-Jan Gubbels / The two type I genotype T. gondii strains, RH, a lab-adapted strain, and GT1, a non-lab-adapted strain, have a genetic difference of only 0.002%, but show remarkable phenotypic differences in vitro. For example, it has long been known that RH’s in vitro virulence (i.e. plaquing capacity) and extracellular survival is far superior to that of GT1, likely due to several decades of adaptation to the in vitro environment (i.e. lab-adaptation). The genetic basis of these phenotypes, however, remains largely unknown despite previous allele-swapping experiments, thus inspiring two hypotheses: 1) epistatic interactions between two or more alleles and/or 2) gene regulatory mechanisms are responsible for lab-adaptive phenotypes. Uncovering the molecular basis underlying lab-adaptive phenotypes will support our growing understanding of T. gondii virulence and suggest therapeutic targets that affect the parasites lytic cycle in a host-independent manner. To answer this question, we applied Evolve and Resequencing (E&R) of GT1 during the first 1500 generations of its lab-adaptation in order to chronologically identify emerging genotype-phenotype correlations. Indeed, lab-adaptation augmented GT1’s in vitro virulence by improving its extracellular survival and reinvasion capabilities- both extracellular phenotypes of the lytic cycle. DNA-sequencing of parallel GT1 populations at multiple evolutionary timepoints (i.e. passages) identified a polymorphic phospholipid flippase gene whose gene expression is critical for in vitro virulence but, unfortunately, the evolved mutations could not be functionally characterized due to technical limitations. RNA-seq of both intracellular and extracellular parasites across several passages identified hundreds of “pro-tachyzoite” differentially expressed genes (DEGs), but only in extracellular parasites, paralleling our phenotypic observations. Interestingly, several upregulated DEGs are connected to fatty acid biosynthesis. Lastly, genetic KO of five seemingly non-related DEGs indicates that GT1’s lab-adaptive in vitro virulence is a complex and polygenic phenotype that is largely controlled by mechanisms independent of genomic mutations. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Vγ9Vδ2 T cells are the main circulating γδ T cells in human adult blood. They are known for their T cell receptor (TCR)-dependent recognition of microbe and endogenous-derived non-peptide pyrophosphate antigens (phosphoantigens, PAg). With the intrinsically biased type 1 immune responses, Vγ9Vδ2 T cells are an important force in the defense of infections and tumors. However, the immune responses of Vγ9Vδ2 T cells in early life infections and in immunotherapies are not clear yet. In this thesis, we explored Vγ9Vδ2 T cell immune responses in both aspects. Vγ9Vδ2 T cells are abundant in human fetal peripheral blood, but compared to their adult counterparts they have a distinct developmental origin, are hyporesponsive towards in vitro phosphoantigen exposure and they do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated in the first part of this thesis their responses upon in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Most congenital infections are caused by viruses, T. gondii is one of the exceptions. The organelle apicoplast present in T. gondii can generate the most potent Vγ9Vδ2 T cell activator. Thus infection in utero with T. gondii makes it a good model to observe Vγ9Vδ2 T cell immune responses in early life. By comparing to age-matched controls, we found that fetal Vγ9Vδ2 T cells were highly expanded in congenital T. gondii infected newborns, and these expanded cells were highly differentiated towards potent cytotoxic effector cells. While the impact of congenital infection on Vγ9Vδ2 T cell expansion and function waned after birth, the Vγ9Vδ2 TCR repertoire of infected infants possessed a clear fetal footprint with public clonotypes, reflecting the Vγ9Vδ2 T cell response in utero. Indeed, verification of the antigen recognition related complementarity-determining region 3 (CDR3) of the TCR for γ and δ chain by high-throughput sequencing revealed the enrichment of three Vδ2 sequences in congenitally-infected infants that are already generated at 8 weeks of gestation. Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. In the second part of this thesis we investigated Vγ9Vδ2 T cell responses towards two kinds of hemiparasite plant Viscum album L. (European mistletoe) extract drugs in vitro. Mistletoe therapy is the most used complementary cancer therapy in European countries. Mistletoe extract drugs are considered to benefit for increasing the quality of life of cancer patients and modulate immune cells, but the mechanism of action is not clear. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe extract drugs by analyzing their functional and TCR responses using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells.In summary, in the first part of this thesis we showed that the human fetus intrinsically possesses a group of Vγ9Vδ2 T cells that are responding to congenital parasite infections that provide potential protective effects to the fetus. In the second part, we provided insight into the in vitro responses of Vγ9Vδ2 T cells towards mistletoe extract drugs, indicating that Vγ9Vδ2 T cells can be an important target in mistletoe therapy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Toxoplasma gondii infection is common in humans and is a significant risk factor for developing the disease schizophrenia. Genetic risk factors are likely required for the disease of schizophrenia to develop. Nurr1 – heterozygous (+/-) mice and wild-type (+/+) mice were evaluated using immune activation of astrocytes within the prefrontal cortex, dopamine levels within the striatum, and measuring the acoustic startle response reaction time by using prepulse inhibition (PPI). T. gondii infected heterozygous (+/-) mice exhibited increased GFAP expression within the prefrontal cortex. Dopamine levels within the striatum were measured and T. gondii infected wild-type (+/+) mice exhibited increased dopamine levels. The acoustic startle response reaction time was measured using PPI and T. gondii infected mice exhibited slower reaction times when compared to controls. These data demonstrate that the Nurr1 (+/-) genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia.

Over the last decade, a significant amount of research has been done on sphingosine-1-phosphate (S1P). S1P regulates survival, proliferation and migration of breast cancer cells45, as well as playing an important but still unknown role in ovarian46, prostate, and glioma47 cell carcinomas. S1P plays a significant role in the regulation, proliferation and angiogenesis, and is therefore an area of considerable interest for cancer research. Previous research in our group has shown that two stilbenoid sphingosine analogs (1 & 2) were effective at sphingosine kinase (SphK) inhibition in addition to showing down-regulation of Vascular Endothelial Growth Factor (VEGF), a novel and distinctly different mechanism of action compared to reported sphingosine kinase inhibitors. Both compounds showed novel toxicity toward the parasite Toxoplasma gondii. T. gondii causes toxoplasmosis; a disease carried by up to 1/3rd of the world’s population and one that has been implicated in schizophrenia. Unfortunately, use of 1 & 2 as fluorescent probes is limited; their emission wavelengths fall between 360 and 380 nm, within the range of background fluorescence. The synthesis of five fluorescent sphingosine analogs is described herein, three of which have emission wavelengths above 420 nm. These compounds show single digit µM cytotoxicity towards T. gondii. The second generation synthesis of Fingolimod (FTY720) is also described. The process was optimized for large-scale production. Improvements to the synthesis included increased atom economy, purification and overall reaction efficiency. Yield was increased to 36%, from 33% in the first generation synthesis. Lastly, a synthesis of p-divinylbenzene is described, using “green” solvents and easy sample work-up. The product contains no meta impurities, an issue that arises when p-divinylbenzene is purchased commercially. / Thesis / Candidate in Philosophy

Indiana University-Purdue University Indianapolis (IUPUI) / Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.