Pleasure is part of hedonic well-being, with roots back to Epicurus 2000 years ago. With the new evolving neuroscientific methods of the late 20th and beginning of the 21st century, we are now able to study the biological components of pleasure. This thesis aims to review empirical studies on the neural correlates of pleasure, which can have important implications for well-being, and treatment of addiction and affective disorders. Recent studies have suggested that pleasure can be separated into coding and causing. Discoveries show that causing of pleasure is created in so called hedonic hot spots, areas of the brain that intensely creates pleasure in the shell of nucleus accumbens and in the ventral pallidum. Areas that codes pleasure on the other hand is represented into more cortical areas of the brain, including orbitofrontal cortex, anterior cingulate cortex and anterior insular cortex. There has been a growing understanding about how pleasure is represented in the brain, and a discussion on interpretations and limitations are provided followed by future research suggestions in the final section.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [63]-80).

Recent bioinformatic analyses identified an operon encoding a potential Type I Secretion System (T1SS) in Treponema pallidum that we hypothesize functions to export key treponemal virulence factors that may contribute to the unique invasiveness and pathogenesis of this spirochete. The membrane fusion protein component (MFP) of T1SSs in other organisms has been shown to play a role in substrate recognition. Hence, the objective of this project is to use the putative MFP, Tp0965, of the potential T. pallidum T1SS to investigate protein-protein interactions with the T. pallidum virulence factor pallilysin (Tp0751) and assess the possibility of the latter being a T1SS substrate. Moreover, protein-protein interactions between Tp0965 and a Treponema phagedenis lysate are investigated with the goal of identifying putative T1SS substrates in this spirochete that could result in the discovery of novel T. pallidum virulence factors via amino acid sequence similarity. Plate-based binding studies and pull-down assays showed a low level of interaction between recombinant Tp0965 and the previously characterized host-component-binding protease, pallilysin, suggesting that the export of this virulence factor could occur via the putative T1SS. Additionally, bioinformatic analyses of the related but cultivable model spirochete T. phagedenis predicted the presence of a potential T1SS homologous to the putative T1SS in T. pallidum. Thus, a more global and unbiased pull-down assay using “bait” Tp0965 and a “prey” T. phagedenis lysate was carried out, followed by mass spectrometric analysis to identify putative novel T1SS substrates with potential homologs in T. pallidum. We successfully identified a T. phagedenis protein, TphBIg, that showed evidence of an interaction with Tp0965. TphBIg seems to possess characteristics of a T1SS substrate suggesting it may be secreted via this system in T. phagedenis. Upon bioinformatic analysis, it was found that TphBIg showed weak amino acid sequence similarity as well as some structural similarity to the T. pallidum protein, Tp0854. Tp0854 is predicted to contain a sialidase and a phosphatase domain with an RTX motif, which is characteristic of some T1SS substrates. Thus, it was hypothesized that if Tp0854 had characteristics of a T1SS, it may interact with Tp0965. Therefore, the phosphatase domain containing the RTX motif was produced recombinantly and plate-based binding studies indeed suggested an interaction with Tp0965, confirming the in silico-predicted interaction. Future experiments to characterize the potential T1SS and substrates in T. pallidum could comprise the functional and structural characterization of the novel putative T1SS substrate, Tp0854. This would include assays to investigate the putative sialidase and phosphatase activities of Tp0854, as well as the identification of Tp0854-Tp0965 interacting sites. Moreover, as a more definite test for T1SS substrate secretion, T. pallidum pallilysin and/or Tp0854 could be expressed heterologously in an E. coli strain harbouring an endogenous T1SS and test for secretion. Similarly, the reconstitution of the T. pallidum putative T1SS in liposomes could be used to further investigate the secretion of pallilysin and/or Tp0854 via this system. Additionally, the optimized unbiased pull-down technique could be further applied to detect more protein-protein interactions within T. pallidum and potentially lead to the identification of more virulence factors that may be secreted via the T1SS. These studies constitute the first investigation of a putative T1SS and substrates within T. pallidum. Thus, insight gained will lead to a better understanding of the mechanisms facilitating T. pallidum host invasion and may reveal new potential vaccine targets to prevent bacterial dissemination and chronic infection. / Graduate

Syfilis är en sexuellt överförbar infektion som orsakas av spiroketen Treponema pallidum subspecies pallidum. Laboratoriediagnostik utgörs i första hand av serologiska test tillsammans med kliniska fynd men på grund av olika faktorer är dessa inte helt pålitliga. Ett flertal olika PCR-metoder utvecklats som påvisar patogenen med hjälp av T-pallidum-specifika gener. En väl studerad gen med hög specificitet är polA-genen som anses vara en mycket robust och känslig metod och lämpar sig väl för att påvisa T. pallidum i kliniska material. Syftet med projektet är att utveckla och optimera en kvalitativ realtids-PCR i singelplex format för verifiering av T. pallidum, för diagnostisering av syfilis. I detta projekt har en realtids-PCR riktad mot polA för detektion av T. pallidum utvecklats. Metodens prestanda utvärderades med en kommersiellt framställd DNA-kontroll med avseende på sensitivitet, specificitet detektions-gräns. När samtliga moment verifierats och fastställts testades metoden på kliniskt material från sår. Den T. pallidum-specifika PCR-analysen visade att det inte förekom korsreaktivitet mot andra agens som förväntas finnas i sår från patient med misstänkt syfilis. Metoden uppvisade en känslighet vid spädning 1:100 (cirka 13 kopior/µl) och en precision på 36,3±0,8 Ct. Det kliniska provet visade på förekomst av T. pallidum dock är känsligheten något sämre än existerande referensmetod. Metoden kan användas i rutindiagnostik av T. pallidum dock bör utfallet från extraktion av DNA från sår studeras ytterligare för att eventuellt öka utbytet och detektera lägre koncentrationer i kliniskt material. / Syphilis is a sexually transmitted infection caused by the spirochete Treponema pallidum subsp. pallidum. Laboratory diagnostics consist primarily of serological tests together with clinical findings for different reasons these methods are not reliable. A variety of PCR methods has been developed that target the pathogen using T. pallidum-specific genes. A well-studied gene with high specificity is the polA gene which is considered very robust and sensitive method and well suited for detection of T. pallidum in clinical materials. The aim of the project is to develop and optimize a qualitative real-time PCR in single-plex format for the verification of T. pallidum, for the diagnosis of syphilis. A real-time PCR targeting polA was developed for detection of T. pallidum. An evaluation of the method's performance was done with a commercially produced DNA control with regards to sensitivity, specificity detection limit and precision. When all test where verified and established, the method was tested on clinical material from ulcers. The results of the T. pallidum-specific PCR-assay showed that there was no cross-reactivity to other agents that are expected to be in ulcers from patients with suspected syphilis. The method showed a sensitivity at dilution 1:100 (about 13 copies/μl) and a precision of 36.3 ± 0.8 Ct.The clinical specimen showed presence of T. pallidum, however, the sensitivity was not as good than the existing reference method. The method can be used in routine diagnostics of T. pallidum, however, the outcome of extraction of DNA from ulcers should be further studied in order to increase the yield and detect lower concentrations in clinical material.

In Parkinson's disease (PD), large parts of the brain transition into states of enhanced neural synchronization. These phase transitions have been associated with the death of dopaminergic neurons as well as with impaired motor function. In this thesis, we address the much-debated question of how parkinsonian synchronization depends on dopamine depletion in the basal ganglia (BG). To this end, we develop spiking neural network (SNN) models of BG circuits and study them via bifurcation analysis. First, we derive mean-field models that allow to account for various forms of short-term plasticity in SNNs. We show that such short-term plasticity mechanisms can lead to highly synchronous, periodic bursting dynamics and discuss the relevance of this bursting regime for PD. Second, we find that the external pallidum, an important part of the BG, cannot cause parkinsonian oscillations autonomously. However, our results suggest that the external pallidum may contribute to the emergence of cross-frequency coupling that has been reported for parkinsonian oscillations. Finally, we describe an open-source Python toolbox that we developed to implement and analyze mean-field models of neural dynamics. Together, this thesis provides insight into BG synchronization processes as well as the mathematical basis and software for future studies of neural synchronization.:1 Introduction 1.1 A complex systems perspective of the brain 1.2 Brain function and the phase transition to synchronized neural activity 1.3 Low-dimensional manifolds of synchronized neural activity 1.4 Phase transitions to synchronized neural activity in Parkinson’s disease 1.5 Thesis overview 2 Mathematical Models and Methods 2.1 A non-linear oscillator model of neural activity 2.2 Dynamical systems methods for the study of neural network models 2.3 Dynamics of a single QIF neuron 3 Low-Dimensional Dynamics in Spiking Neural Networks 3.1 Mean-field approaches in neuroscience 3.2 Dynamics of QIF networks with post-synaptic STP 3.3 Dynamics of QIF networks with spike-frequency adaptation 3.4 Mean-field dynamics of QIF networks with pre-synaptic STP 3.5 Discussion 4 Phase Transitions and Neural Synchronization in the External Pallidum 4.1 A new perspective on GPe structure and function 4.2 GPe model definition and analysis 4.3 Phase transitions in the GPe under static and periodic input 4.4 Discussion 5. Modeling of Neural Mean-Field Dynamics Via PyRates 5.1 Computational modeling in neuroscience 5.2 The Framework 5.3 Pre-implemented methods for neural modeling workflows 5.4 Results 5.5 Discussion 6. Conclusion and Outlook

This diploma thesis is dealing with methods of identification genetic variability in sequencing data. The resarch is targeted to bacterial strains of Treponema pallidum. The sequencing was performed by Illumina platform. There is a proposition of method to identificate variable spots in resequenced genomes and their analysis and comparation across all processed genomes.

No / Aim: A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. Materials & methods: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. Results & conclusion: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.

Le globus pallidus (GP) des primates reçoit une innervation massive des neurones GABAergiques du striatum qui co-libèrent la substance P (SP). Afin d'approfondir notre connaissance de l'interaction de la SP au niveau pallidal, nous avons étudié la localisation cellulaire et subcellulaire de ce peptide et de ses récepteurs à hautes affinités neurokinine-1 (NK-lR) et neurokinine-3 (NK-3R) dans le GP de singes écureuils. Un grand nombre de neurones et de fibres dans le segment externe (GPe) et interne (GPi) du GP exprimaient de l'immunoréactivité pour NK-lR ou NK-3R en position pré- et postsynaptique. Les NK-lR et NK-3R étaient principalement localisés dans le cytoplasme ou sur la membrane plasmique, mais en dehors des jonctions synaptiques. Certaines terminaisons axonaies immunoréactives pour la SP exprimaient préférentiellement NK-3R. Ces données suggèrent que la SP peut influencer de manière significative le traitement de l'information neuronale voyageant à travers les ganglions de la base. / The primate globus pallidus (GP) receives a massive innervation from GABAergic striatal neurons that co-release substance P (SP). We used single and double antigen staining retrieval methods to study cellular and subcellular localization of SP and its high affinity receptors NK-IR and NK-3R in GP of squirrel monkeys. A large number of neurons and fibers in GPe and GPi expressed NK-IR or NK-3R. NK-IR and NK-3R were mainly associated with intracellular sites or located at extrasynaptic positions on the plasma membrane. SP+ axon terminals preferentially expressed NK-3R. Distribution of NK-IR and NK-3R indicates that SP effects at pallidal level are mediated through postsynaptic receptor as well as presynaptic autoreceptors and heteroreceptors. These data suggest that SP may influence in a significant manner the treatment of neural information that flows through the basal ganglia.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2016-2017 / L’élucidation de la position qu’occupent les projections sérotoninergique (5-HT), cholinergique (ACh) et dopaminergique (DA) du tronc cérébral dans l’organisation anatomofonctionelle du globus pallidus externe (GPe) et interne (GPi) au sein des ganglions de la base chez le primate est primordiale à la compréhension de ce système neuronal hautement complexe impliqué dans le contrôle du comportement moteur. Les travaux de recherche consolidés dans la présente thèse rapportent les résultats principalement obtenus chez le singe écureuil (Saimiri sciureus) à l’aide de marquages immunohistochimiques et de quantifications stéréologiques servant à évaluer la distribution régionale et les caractéristiques ultrastructurales des varicosités axonales 5-HT, ACh et DA observées dans le pallidum. Nos données ont permis l’éloboration d’un nouveau modèle du neurone pallidal en tenant compte de la hiérarchie et des caractéristiques neurochimiques de ses entrées synaptiques. Ainsi, l’analyse quantitative en microscopie optique révèle que le GPe et le GPi reçoivent des innervations 5-HT, ACh et DA de densités variables et distribuées de façon hétérogène. Plus particulièrement, le GPe est innervé par 600 000 varicosités 5-HT/mm3 de tissu, 500 000 varicosités ACh/mm3 et 170 000 varicosités DA/mm3. En revanche, le GPi reçoit 600 000 varicosités 5-HT/mm3, 250 000 varicosités ACh/mm3 et 190 000 varicosités DA/mm3. De plus, la 5-HT, l’ACh et la DA ciblent préférentiellement les secteurs correspondant aux territoires fonctionnels associatifs et limbiques du pallidum, suggérant un rôle de ces projections dans la planification du comportement moteur ainsi que dans la régulation de l’attention et de l’humeur. Nos analyses en microscopie électronique révèlent que très peu de ces varicosités axonales établissent un contact synaptique, puisque plus de 70% des varicosités 5-HT, ACh et DA sont complètement dépouvues de jonction synaptique. Ainsi, bien que la 5-HT, l’ACh et la DA seraient en mesure de moduler directement les neurones pallidaux grâce à la transmission synaptique, leur plus grande influence s’opérerait par la transmission volumique, permettant d’influencer à la fois les neurones pallidaux et leurs afférences, principalement du striatum et noyau subthalamique. L’ensemble de ces résultats indique que les projections 5-HT, ACh et DA du tronc cérébral agissent de concert avec les afférences plus robustes en provenance du striatum et du noyau subthalamique. Ces nouvelles données neuroanatomiques positionnent le tronc cérébral en tant qu’acteur important dans l’organisation anatomique et fonctionnelle du pallidum chez le primate et doivent être prises en considération dans l’élaboration de nouvelles approches thérapeutiques visant à contrer les processus neurodégénératifs qui affectent les ganglions de la base, tel que la maladie de Parkinson. / A better understanding of the place that the brainstem serotoninergic (5-HT), cholinergic (ACh) and dopaminergic (DA) projections occupy in the anatomical and functional organization of the primate external (GPe) and internal (GPi) globus pallidus within the basal ganglia is primordial to enhance our comprehension of this complex neuronal system involved in the control of motor behaviors. The present thesis reports novel neuroanatomical findings gathered in the squirrel monkey (Saimiri sciureus) using immunohistochemical labelings and the stereological quantification approach to determine the distribution and ultrastructural features of the 5-HT, ACh and DA axon varicosities observed in the monkey pallidum. Our findings have led to the elaboration of a new model of the pallidal neuron based on a precise knowledge of the hierarchy and neurochemical features of its various synaptic inputs. Quantitative analyses at the light microscopic level reveal that the GPe and GPi receive heterogeneously distributed 5-HT, ACh and DA innervations in variable densities. More precisely, the GPe is innervated by 600,000 5-HT varicosities/mm3 of tissue, 500,000 ACh varicosities/mm3 and 170,000 DA varicosities/mm3. In contrast, the GPi receives 600,000 5-HT varicosities/mm3, 250,000 ACh varicosities/mm3 and 190,000 DA varicosities/mm3. Furthermore, the 5-HT, ACh and DA innervations preferentially target sectors corresponding to the associative and limbic pallidal functional territories, suggesting that these brainstem inputs are involved in the planification of motor behavior, more than in its execution, and in the regulation of attention and mood. Electron microscopic analyses reveal that very few of these axon varicosities establish genuine synaptic contacts, since more than 70% of these axon varicosities are devoid of any synaptic junction. Hence, even though the 5-HT, ACh and DA innervations can directly modulate pallidal neurons through synaptic delivery, the vast majority use volume transmission to influence both pallidal neurons and their major afferents from the striatum and the subthalamic nucleus. Altogether, these results indicate that the 5-HT, ACh and DA projections act in concert with the more robust striatopallidal and subthalamopallidal inputs. Our novel neuroanatomical data suggest that these brainstem projections are ideally positioned to act as major modulators of the primate globus pallidus. The understanding of the relation between the brainstem and the basal ganglia is a prerequisite for the development of new therapeutics avenues for the treatment of neurodegenerative disorders involving the basal ganglia network, such as Parkinson’s disease.