>Magister Scientiae - MSc / The high-spin states of the nucleus 152Gd have been populated via the 152Sm (α, 4n) 152Gd fusion-evaporation reaction at a beam energy of 45MeV. The emitted γ rays were observed with the AFRODITE spectrometer array. The previously known decay scheme of 152Gd was extended. Directional Correlation from Oriented states of nuclei (DCO) and linear polarization measurements were performed in order to assign spins and parities in the 152Gd decay scheme. An alternative interpretation of the first excited Kπ = 0+ band, previously considered to be a β-vibration is discussed.

Unlike in Classical Arabic, this study hypothesized that word-initial consonant clusters exist in Najdi Arabic as a result of first vowel deletion. The goal of this study was to investigate the word-initial consonant cluster patterns of Najdi Arabic and measure the sonority scale of this particular position. Ten native Najdi Arabic speakers were asked to pronounce 24 words and 24 sentences that contained all the possible consonant cluster patterns that could occur in Najdi Arabic. The output of the subjects revealed that Najdi Arabic does have initial consonant clusters in certain environments and that the minimum sonority distance was one step between the first and second onsets. The study found that the sonority distance between the first and second onsets plays a role in forming initial consonant clusters in Najdi. Additionally, the existence of less-marked consonant clusters was found to be more frequent than the more marked ones. Finally, the study proposed examining the pattern of the deleted vowel in future studies to determine whether it plays a role in the results.

The caudal nucleus tractus solitarius (cNTS) is the key recipient of the primary afferents from visceral sensory neurons and also an important site that processes and integrates gastrointestinal, cardiovascular and respiratory functions. Glutamate and gamma-aminobutyric acid are the major neurotransmitters within the NTS, but studies have suggested that nicotinic acetylcholine receptors (nAChRs) and transient receptor potential (TRP) channels can modulate excitatory/inhibitory neurotransmission. I have designed studies to understand the role of nAChRs and TRP channels in the modulation of neurotransmission in the cNTS. In the first aim, experiments were designed to test the hypothesis that the cNTS contains function-specific subsets of neurons whose responsiveness to nicotine correlates with the target of their axonal projections. cNTS neurons send axonal projections to brain regions such as parabrachial nucleus (PBN), hypothalamic paraventricular nucleus (PVN), nucleus ambiguous (NA), dorsal motor nucleus of the vagus (DMV) and the caudal ventrolateral medulla (CVLM) and are involved in integrating autonomic and neuroendocrine functions. Presynaptic/postsynaptic modulation by nAChRs differ in the axonal projections of cNTS neurons, studying of which would provide better understanding of this complex integration. In vivo fluorescent tracing combined with in vitro slice patch-clamp electrophysiological recordings from anatomically identified caudal NTS neurons were used to study the expression and function of nAChRs (mainly á3â4 containing nAChRs) in the cNTS. Results from these studies demonstrate that presynaptic and postsynaptic responsiveness of caudal NTS neurons to nicotine correlates with the areas the neurons project to in the following order of prevalence: DMV>PVN>NA>CVLM>PBN (for presynaptic responses) and DMV>CVLM>PBN>NA>PVN (for postsynaptic responses). In the second aim, experiments were designed to test the hypothesis that nociceptive TRP channels TRPV1 (vanilloid) and TRPA1 (ankyrin) modulate synaptic transmission in the NTS. As a result of this modulation, the efferent functions that control autonomic and visceral functions will be regulated and account for the changes in autonomic neuropathy as patients with diabetes develop significant alterations in blood pressure and heart rate as well as silent myocardial ischemia as a result of blunted pain carrying ability. Results obtained from these studies demonstrated that TRPV1 and TRPA1 mRNA were detected in the dorsal root ganglion (DRG), but not in the NTS. Immunofluorescence studies revealed that TRPV1 and TRPA1 were expressed in the solitary tract central sensory terminals inputs to NTS but not in NTS neurons. This suggests that TRPV1 and TRPA1 are expressed only in solitary tract. Administration of capsaicin (TRPV1 agonist) and allyl isothiocyanate (AITC, TRPA1 agonist) both increased the frequency of s/mEPSCs without affecting spontaneous and miniature inhibitory postsynaptic currents (s/mIPSCs). Next, the modulation of TRPV1- and TRPA1-induced responses by utilizing a PKC activator (PDBu) was examined. Incubation of slices with PDBu synergistically increased the mEPSC frequency following capsaicin application suggesting an increased receptor affinity; however following application of AITC there was no significant change, suggesting that activation by covalent modification does not enhance binding affinity. Finally, the specificity of TRPV1 and TRPA1 effect on synaptic transmission by ablating TRPV1 and TRPA1were tested. There was no modulation of synaptic transmission in these animals, further confirming that capsaicin- and AITC-mediated modulation of synaptic transmission are specifically mediated by TRPV1 and TRPA1, respectively. Furthermore, animals with painful diabetic peripheral neuropthy exhibited enhanced synaptic activity at the NTS, suggesting a role in nociception and other visceral functions. In summary, nAChRs, TRPV1 and TRPA1 are expressed in the NTS and activation of which modulate excitatory synaptic transmission. The results obtained from these studies and their interpretation may provide a better understanding of the central mechanism of modulation on efferent functions from NTS that regulate cardiovascular, respiratory and gastrointestinal functions.

Mammalian cells are known to respond to both extra- and intra- cellular forces as well as the physical properties of the surrounding tissue. There is increasing evidence to support the fundamental role of force, applied to or generated within cells, in maintaining proper tissue function. The mechanical integration from the exterior of a cell to the interior of the nucleus is crucial for cellular sensing of, and response to, the physical environment. Further, misregulation of this mechanosensitive ability can lead to the development or propagation of many diseases such as cancers, cardiovascular diseases, and tissue fibrosis. In this thesis, we investigate the role of various proteins in regulating the mechanical properties of mammalian cells. We also develop techniques to examine the propagation of forces through cells and multicell systems with the aim of elucidating critical biophysical factors involved in regulating cell function. The idea that the genome can be regulated through changes in forces applied to cells or changes in the propagation of forces through a cell, (i.e. mechanotransduction) is becoming widely accepted. The complex interplay between biochemical and biophysical mechanisms that ultimately control mechanotransduction are beginning to be uncovered; however, a true understanding of this remarkable cellular process has not yet been achieved. By investigating multiple factors which impact mechanosensitivity (such as protein expression, cell-cell and cell-environment connections, cell generated contractions, and physical connections through the cellular interior), we aim to further the understanding of potential pathways of mechanotransduction. Through novel studies and technological advances, the field of cellular biomechanics will continue to grow as we hope to uncover the physical mechanisms that regulate cell function or lead to disease.

Les arguments en faveur d’un rôle important de l'architecture des chromosomes en interphase pour la régulation des gènes et la maintenance du génome s’accumulent rapidement. Au cours de l'interphase, les chromosomes sont positionnés de façon non aléatoire l’un par rapport à l'autre et fournissent ainsi des points de repère nucléaires. Deux types d'interactions contribuent probablement à ce positionnement non aléatoire: (i) des domaines subchromosomiques interagissent avec des structures nucléaires telles l'enveloppe nucléaire (EN) et (ii) des interactions intrachromosomiques s’établissent entre des loci situés de façon linéairement distante en cis sur un même chromosome. Contribuant à l’expansion de ce domaine de recherche, nous avons poursuivi le développement d’une technique préalablement établie au laboratoire pour détecter des interactions protéine-protéine. Le développement de cette technique nouvelle a constitué une part de ce travail de thèse accompli sur des cellules humaines. Elle se base sur le marquage par la biotine de composants de la chromatine qui en interphase se trouvent à proximité immédiate de l’EN. Les cellules ont été traitées pour exprimer (i) la biotine ligase BirA fusionnée à l’émerine, une protéine de l’EN, conjointement avec (ii) une variante d’histone, l’histone macroH2A, en fusion avec un peptide accepteur de biotine. L'étiquette biotine déposée sur l’histone macroH2A pendant l'interphase est ensuite détectée par microscopie à fluorescence sur des cellules en mitose étalées sur lames. Les chromosomes mitotiques marqués peuvent en outre être caractérisés par des techniques plus classiques de caryotypage. Nous avons nommé cette technique «topokaryotypage» car elle peut fournir des informations d’ordre à la fois topologique et caryotypique. Son développement pas à pas a nécessité la production d'une lignée cellulaire ad hoc et une optimisation fine du protocole. Ce travail de thèse peut déboucher sur des questions biologiques explorées sur cellules uniques. A titre d’application, une analyse comparative a été réalisée par topokaryotypage sur des cellules cultivées in vitro dans diverses conditions de stress expérimentales. L’utilisation du topocaryotypage pourrait fournir des informations précieuses sur les mécanismes à la base de l’organisation et de la dynamtique des noyaux cellulaires. / Evidence is rapidly accumulating that the architecture of interphase chromosomes is important for both gene regulation and genome maintenance. During interphase, chromosomes are nonrandomly positioned with respect to each other and thus they provide nuclear landmarks. Two kinds of interactions are likely to contribute to this nonrandom positioning: (i) subchromosomal domains interact with nuclear structures such as the nuclear envelope (NE) and ii) intrachromosomal interactions take place between linearly distant loci positioned in cis on the same chromosome. As a contribution to this expanding research domain, we have built upon an existing approach previously established in the laboratory to detect protein-protein interactions. The new technique was developed in human cells as part of the present PhD research. It is based on biotin labelling of chromatin components which are in close proximity with the nuclear envelope (NE) in interphase cells. Cells were made to express (i) the biotin ligase BirA fused to the NE protein emerin together with (ii) a fusion between a biotin acceptor peptide and macroH2A, a variant core histone. The biotin label deposited on the macroH2A histone during interphase is then detected by fluorescence microscopy on mitotic cells spread on slides. The biotin-labelled mitotic chromosomes can be further characterized using more classical karyotyping techniques. We refer to this new technique as “Topokaryotyping” since it can provide both topological and karyotypic information. Its step-by-step development has required the establishment of an ad hoc cell line and a fine protocol optimization. This PhD work could pave the way for biological questions explored at a single cell level. As an illustration, a comparative topokaryotyping analysis was performed on cells cultivated in vitro in various experimental stress conditions. It is envisioned that using this technique can provide valuable mechanistic insights relevant to the organization and dynamics of cell nuclei.

Distribution of orexin-A-like immunoreactivity (ORX-LI) in rat brains was investigated with the use of a rabbit polyclonal antibody against the full length peptide orexin A. Virtually all the ORX-LI cell bodies were observed in the lateral hypothalamus at the level of median eminence. The large majority of ORX-LI neurons appeared spherical or fusiform, 20-30 μm in diameter and issued two to five cell processes with few secondary branchings. Numerous ORX-LI fibers were observed in subregions of the hypothalamus. ORX- LI cell processes were sparsely distributed in the cortex, hippocampus and thalamus. Many varicose ORX-LI cell processes were situated close to the 3rd and lateral ventricles, some of which appeared to be protruding into the lumen. As a corollary, orexin A may be released into the ventricles and interact with neurons in distant targets, in addition to influencing the activity of neurons with which ORX-LI axons make synaptic contacts.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20973号 / 医博第4319号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 溝脇 尚志, 教授 村井 俊哉, 教授 鈴木 実 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In addition to bradykinesia and tremor, patients with Parkinson’s disease (PD) are known to exhibit non-motor symptoms such as apathy and hypomimia but also impulsivity in response to dopaminergic replacement therapy. Moreover, a plethora of studies observe differences in electrocortical and autonomic responses to both visual and acoustic affective stimuli in PD subjects compared to healthy controls. This suggests that the basal ganglia (BG), as well as the hyperdirect pathway and BG thalamocortical circuits, are involved in affective processing. Recent studies have shown valence and dopamine-dependent changes in synchronization in the subthalamic nucleus (STN) in PD patients during affective tasks. This thesis investigates the role of dopamine, valence, and laterality in STN electrophysiology by analyzing event-related potentials (ERP), synchronization, and inter-hemispheric STN connectivity. STN recordings were obtained from PD patients with chronically implanted electrodes for deep brain stimulation during a passive affective picture presentation task. The STN exhibited valence-dependent ERP latencies and lateralized ‘high beta’ (28–40 Hz) event-related desynchronization. This thesis also examines the role of dopamine, valence, and laterality on STN functional connectivity with the anterior cingulate cortex (ACC) and the amygdala. The activity of these limbic structures was reconstructed using simultaneously recorded electroencephalographic signals. While the STN was found to establish early coupling with both structures, STN-ACC coupling in the ‘alpha’ range (7–11 Hz) and uncoupling in the ‘low beta’ range (14–21 Hz) were lateralized. Lateralization was also observed at the level of synchrony in both reconstructed sources and for ACC ERP amplitude, whereas dopamine modulated ERP latency in the amygdala. These results may deepen our current understanding of the STN as a limbic node within larger emotional-motor networks in the brain. / Neben Bradykinese und Tremor weisen Patienten mit Morbus Parkinson (PD) bekannterweise nicht-motorische Symptome auf wie Apathie und Hypomimie, aber auch Impulsivität, welche durch Dopaminersatztherapien bedingt ist. Viele Studien belegen außerdem Unterschiede von kortikalen und autonomen Reaktionen auf sowohl visuelle als auch akustische Reize bei Patienten mit PD im Vergleich zu gesunden Kontrollgruppen. Dies legt nahe, dass sich die Basalganglien (BG), und auch die hyperdirekte Verbindung sowie die BG-thalamokortikalen Schleifen, an der Affektbearbeitung beteiligen. Jüngere Studien haben Valenz- und Dopamin-bedingte Veränderungen der Synchronisierung im Nucleus subthalamicus (STN) von Parkinson-Patienten bei affektiven Aufgaben belegt. Diese Promotionsarbeit untersucht die Rolle von Dopamin, Valenz und Lateralität in der STN-Elektrophysiologie mittels Analysen von ereigniskorrelierten Potentialen (ERP), Synchronisierung und interhemisphärischer funktioneller Konnektivität. STN-Aufzeichnungen wurden von Patienten mit dauerhaft implantierten Elektroden für die Tiefenhirnstimulation während einer passiven Aufgabe abgeleitet, bei den ihnen Bilder mit emotionalen Inhalten gezeigt wurden. Der STN wies Valenz-bedingte ERP-Latenz und lateralisierte ereigniskorrelierte Desynchronisierung in ‘hohem Beta’ (28–40 Hz) auf. Diese Dissertation untersucht auch die Rolle von Dopamin, Valenz und Lateralität bezüglich der funktionellen Konnektivität zwischen dem STN und dem Gyrus cinguli pars anterior (ACC) sowie der Amygdala. Die Aktivität dieser Strukturen wurde aus simultanen elektroenzephalographischen Aufzeichnungen rekonstruiert. Obwohl eine STN-Kopplung mit beiden Strukturen auftritt, war die STN-ACC-Kopplung im ‘Alpha’- Bereich (7–11 Hz) und die Entkopplung im ‘niedrigen Beta’-Bereich (14–21 Hz) lateralisiert. Lateralisierung wurde auch an der Synchronisierung in beiden rekonstruierten Quellen und an der ACC-ERP-Amplitude festgestellt, wohingegen Dopamin die ERP-Latenz in der Amygdala modulierte. Diese Ergebnisse mögen das gegenwärtige Wissen vom STN als limbischem Knoten innerhalb größerer affektiv-motorischer Schleifen im Gehirn vertiefen.

Hypertension (HTN) is a critical public health issue estimated to contribute to 10% of deaths worldwide. Additionally, the salt sensitivity of blood pressure, an exaggerated pressor response to elevated dietary sodium intake, is estimated to be present in approximately 50% of the hypertensive population and 25% of the normotensive population. This is a critical problem as the average American consumes roughly three times the daily sodium intake recommended by the American Heart Association. Our laboratory has previously identified a critical role of Hypothalamic Paraventricular Nucleus (PVN) Gαi2 proteins in the maintenance of salt resistance and normotension in the rat. Salt resistant rats such as the Sprague-Dawley (SD) rat site- specifically upregulate these proteins in response to elevated dietary sodium intake to facilitate sympathoinhibition, natriuresis, and normotension. In contrast, in the Dahl Salt Sensitive (DSS) rat, and in salt resistant rats in which this protein is experimentally downregulated, our laboratory has identified the development of renal nerve-dependent sympathoexcitation and salt-sensitive hypertension (ssHTN). However, the neural mechanisms whereby PVN Gαi2 proteins facilitate salt resistance are unclear. In addition, there is a robust literature in other rat models of HTN suggesting that both neuroinflammation in the PVN as well as an imbalance between PVN inhibitory GABAergic and excitatory glutamatergic signaling contribute to elevations in sympathetic outflow to promote HTN. In this study, SD rats infused chronically with either targeted Gαi2 oligodeoxynucleotides (ODNs) or control scrambled (SCR) ODNs and challenged with either normal (0.6% NaCl) or high-salt (4% NaCl) diets were used to demonstrate that 1) PVN microglial activation and associated pro-inflammatory cytokine production contribute to the development of Gαi2 protein dependent ssHTN, 2) sex-dependent PVN microglial-mediated neuroinflammation precedes and likely drives the development of sympathoexcitation following high dietary sodium administration in male but not female Gαi2 protein dependent ssHTN, and 3) PVN GABAergic and glutamatergic signaling is disrupted and imbalanced, favoring excitation over inhibition, following elevated dietary sodium intake in Gαi2 protein dependent ssHTN. Together, these findings shed light on the pathological neural processes that occur in the absence of PVN Gαi2 protein upregulation and reveal potential mechanistic targets in the management of ssHTN.

Thesis advisor: David R. Burgess / Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.