In the presented research we propose to improve on historically accepted radiobiological models via Monte Carlo simulation of radiation tracks passing through a cell nucleus modeled with up-to-date subnuclear structures. This is performed by generating a radiation track database using the Monte Carlo code, Geant4-DNA, that simulates radiation interactions at the nanometer scale of DNA. These tracks are called upon from the database and intersected with a cell nucleus model that incorporates DNA-containing structures. This allows for a Monte Carlo simulation of how DNA double strand breaks are produced by radiation. The results can be used to correlate to many experimentally observed biological endpoints, e.g. chromosome aberrations as well as cell death.

The circumnuclear disc (CND) orbiting the Galaxy's central black hole is a reservoir of material that can ultimately provide energy through accretion, or form stars in the presence of the black hole, as evidenced by the stellar cluster that is presently located at the CND's centre. In this paper, we report the results of a computational study of the dynamics of the CND. The results lead us to question two paradigms that are prevalent in previous research on the Galactic Centre. The first is that the disc's inner cavity is maintained by the interaction of the central stellar cluster's strong winds with the disc's inner rim, and secondly, that the presence of unstable clumps in the disc implies that the CND is a transient feature. Our simulations show that, in the absence of a magnetic field, the interaction of the wind with the inner disc rim actually leads to a filling of the inner cavity within a few orbital time-scales, contrary to previous expectations. However, including the effects of magnetic fields stabilizes the inner disc rim against rapid inward migration. Furthermore, this interaction causes instabilities that continuously create clumps that are individually unstable against tidal shearing. Thus the occurrence of such unstable clumps does not necessarily mean that the disc is itself a transient phenomenon. The next steps in this investigation are to explore the effect of the magnetorotational instability on the disc evolution and to test whether the results presented here persist for longer time-scales than those considered here.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Previous research has suggested that lobeline, a plant alkaloid derived from Lobelia inflate, has potential to be an efficacious pharmacotherapy for the treatment of methamphetamine dependence. In addition to attenuating methamphetamineinduced dopaminergic alterations in vitro, lobeline has been shown to decrease the primary rewarding effects and discriminative stimulus properties of methamphetamine in rats. It is of clinical interest to assess the utility of lobeline to decrease methamphetamine conditioned cues as these cues have been shown to significantly contribute to relapse. The current studies assessed the ability of lobeline to block the acquisition and expression of methamphetamine-induced conditioned place preference in rats. Lobeline blocked the acquisition of methamphetamine-induced conditioned place preference when a low dose of methamphetamine was used during conditioning. However, this blockade was surmounted with higher doses of methamphetamine. Furthermore, the expression of methamphetamine-induced conditioned place preference is attenuated following repeated administration, indicating that lobeline not only blocks the primary reinforcing effects of methamphetamine, but it also blocks the environmental cues that become associated with drug administration. These results provide further evidence that lobeline may be an efficacious treatment for methamphetamine dependence. The rewarding properties of psychostimulants are thought to be mediated, at least in part, by the nucleus accumbens shell. The effects of lobeline on methamphetamine-induced alterations in this dopaminergic region were assessed using microdialysis in rats. Acute lobeline did not have an effect on the methamphetamine-induced increases in dopamine, indicating that repeated lobeline administration may be more efficacious. Interestingly, lobeline potentiated the methamphetamine-induced decrease of the dopamine metabolite, DOPAC. These results suggest that acute lobeline may function to redistribute vesicular dopamine pools within the terminal bouton.

Stress is known to impair spatial navigation in rat models of declarative memory, and declarative memory in humans, but the effects on spatial navigation in humans are unclear. At least four models have been proposed to account for the cognitive effects of stress, based on the two different physiological stress response systems (the sympathetic-adrenal-medullary (SAM) and the hypothalamic-pituitary-adrenal (HPA) systems) and the effects of these responses on the hippocampus and (sometimes) other subcortical structures. In this dissertation, I examined the effects of an acute (experimental) stressor on human spatial navigation in three variations of virtual Morris water mazes designed to dissociate between hippocampus-dependent (allocentric) and hippocampus-independent (egocentric) forms of navigation. Results were considered in the light of all 4 models. Experiment 1 used a dual-strategy Morris water maze to test whether acute stress influences navigational strategy selection and whether this effect is mediated by the activation of the HPA or the SAM system. Surprisingly, stress increased hippocampus-based strategy selection, and did so in the presence of SAM but not HPA activation. Experiment 2 used new dual-strategy and place mazes to examine the effects of acute stress on both strategy selection and allocentric navigational performance. It also attempted to contrast the effects of stress at a short delay, which would favour mediation by the SAM system, and a longer, 30 minute delay (from stressor onset), which would favour mediation by the HPA system. Contrary to expectations, results revealed no effect of stress when tested immediately and sex-dependent impairments of performance (in females) and allocentric strategy selection (in males) at the delay. Experiment 3 used the same mazes as Experiment 2, plus a new cue maze to examine the effects of acute stress on strategy selection and both allocentric and egocentric navigational performance after a 30 minute delay. Results confirmed that stress reduces allocentric strategy selection and impairs allocentric performance, but also has sex-dependent effects on egocentric performance: in females, stress enhanced navigation (as expected) but in males, stress impaired it. None of the 4 models provided a good explanation for these results, suggesting that current accounts of the cognitive effects of stress may be inadequate. / Graduate

The nucleus circularis has been linked to a role in regulating osmotic thirst but evidence has also shown that full bilateral destruction of the nucleus circularis was not necessary to achieve a deficit in drinking behavior after an osmotic challenge. The present study attempted to answer two primary research questions. The first question was whether osmoreceptive cells existed around the nucleus circularis in a homogeneous fashion or if these cells existed in a structured fashion stretching from the nucleus circularis forward. The second question was whether animals with lesions of the nucleus circularis and the surrounding areas were different in normal daily water intake than animals with no lesions. The first question was approached by lesioning the nucleus circularis, the area one millimeter anterior to the nucleus circularis, one millimeter posterior to the nucleus circularis, one half of a millimeter medial to the nucleus circularis and using a sham group which had the electrode passed through the brain to a spot one millimeter above the nucleus circularis but passing no current. All animals were then given an osmotic challenge which consisted of half of each group with an injection of hypertonic saline while the other half of each group was given isotonic saline. After a five-day recovery period, the injection procedure was reversed. Water consumption on each test day was measured at ten-minute intervals for one hour. Difference scores were then computed by subtracting the amount of water consumed after hypertonic saline injection from the amount of water consumed after isotonic saline injection. The difference scores were then used in an analysis of variance which revealed a significant difference between groups. A subsequent post hoc test showed that the nucleus circularis group was different from all other groups except for the anterior lesion group which showed a trend in the same direction as the nucleus circularis group. The second research question was approached in two ways. The first way was to simply record the amount of water consumed in each twenty-four hour period. An analysis of variance showed no significant difference between any of the groups. The second method for testing the second research question was to put the animals on a twenty-three hour water deprivation schedule and measure the amount of water consumed during the one hour when water was available. Once again, no significant differences were observed.

Chronic pain alters sensory responses and carries a strong emotional component. Persistent pain can heighten pain experiences, resulting in hyperalgesia and allodynia. Further, patients suffering from chronic pain are more prone to experience a range of affective disorders including depression, sleep dysregulation, panic disorders, anxiety abnormalities and stress-related disorders including post-traumatic stress disorder (PTSD). Hence while pain serves a protective function to prevent additional physiological harm by driving behavioral and cognitive responses, chronic or persistent pain can lead to maladaptive nociceptive responses and exacerbate psychopathologies. Among brain regions, the amygdala is centrally situated to integrate the many descending and ascending signals to modulate the sensory and emotional components of pain. The amygdala is well studied for its role in fear and stress-related behavioral processes. The central nucleus of the amygdala (CeA), and in particular the lateral capsular subdivision of the CeA (CeLC), receives prominent ascending pain neurotransmission via the spino- parabrachioamygdaloid tract. In this pathway, peripheral nociceptive signals carried via primary sensory Aδ- and C-fibers terminate in the dorsal horn where second order neurons send projections via the spino-parabrachial pathway to the lateral parabrachial nucleus (LPBn). Thus, the LPBn collects cutaneous (mechanical and thermal), deep (muscular and articular) and visceral nociceptive signals and relays the information in a highly organized manner principally to the CeLC for nociceptive processing. In pain, the CeA and the LPBn-CeLC projections have been shown to undergo plasticity in the forms of enhanced synaptic transmission and alterations in neurotransmitter and receptor expression. Accordingly, the neurocircuit intersections in the CeA can modulate the sensory and emotional responses to pain. Yet despite these associations, the mediators and mechanisms underlying the emotional consequences of pain are poorly understood. Pituitary adenylate cyclase activating polypeptide (PACAP) is a neural and endocrine pleiotropic peptide important in the development and homeostatic regulation of many physiological systems. Recently, the expression of PACAP and its cognate PAC1 receptor has been shown to be upregulated in specific limbic regions by chronic stress. PACAP infusions into several limbic regions is anxiogenic, and altered blood PACAP levels and PAC1 receptor polymorphism have been associated with PTSD and other stress-related disorders. Here, we establish that CeLC PACAP originates from the LPBn as part of the spino-parabrachoamygdaloid pathway. Chronic pain enhanced PACAP expression along LPBn-CeLC projections, indicating it may be a component of pain- related plasticity. CeA PACAP signaling was sufficient to induce nociceptive hypersensitivity and anxiety-like behaviors. In a chronic neuropathic pain model, CeA PACAP signaling was found to contribute to heightened anxiety-like behaviors and nociceptive responses. Further, we characterized one prominent intracellular signaling mechanism through which CeA PACAP signaling influences these behaviors. In these experiments we provide evidence that CeA PACAP signaling plays an important role in the emotional components of pain and that alterations in CeA PACAP signaling are part of pain-related plasticity. This work establishes novel molecular mechanisms that underlie the emotional component of pain and may contribute to the development of chronic pain and associated affective disorders.

In the cell we can find a lot of small noncoding RNAs, which are important for many processes. Among those RNAs are small nuclear RNA uridin rich, which with proteins create U snRNP.These particles play important role in pre-mRNA splicing. In this process are noncoding sequences (introns) removed and coding sequences (exons) are joined. It is catalyzed by spliceosome. The core of this spliceosome is created by U1, U2, U4, U5 and U6 snRNP. They are essential for this process. Some steps of U snRNP biogenesis proceed in nuclear structures called Cajal bodies (CB). In my thesis I focused on factors, which are important for targeting U snRNA into CB. I used U2 snRNA like a model. With the aid of microinjection of fluorescently labeled U2 snRNA mutants I found, that the Sm binding site on U2 snRNA is essential for targeting to CB. Knock down of Sm B/B'showed us, that Sm proteins are necessary for transport U2 snRNA to CB. Sm proteins are formed on U2 snRNA by SMN complex. Deletion of SMN binding site on U2 snRNA had the same inhibition effect. From these results we can see, that Sm proteins and SMN complex are important for U2 snRNA biogenesis espacially for targeting into CB. Key words: U snRNP, Cajal body, U snRNA, cell nucleus

Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...

Giardia intestinalis is a parasitic protist that causes one of the most common diarrheal disease of parasite origin. The cell of Giardia contains two nuclei with unknown number of chromosomes until recently. Karyotype was determined five years ago using conventional cytogenetic method by Tůmová and collaborators. In my work, I assessed karyotype of four isolates, six lines and three clonal lines by the same method. It was confirmed, that two nuclei within one cell could differ in chromosome number, the differences found were 1, 2 or 6 chromosomes. Aneuploid number of chromosomes was found too. In case that both nuclei within single cell contained the same number of chromosomes, there were 10 chromosomes indentified in each nucleus. It was also revealed, that karyotype is not specific feature for different genetic groups (in this work assemblages A and E). Karyotype can be different even among lines and clonal populations derived from the same isolate. Changes in karyotype in the course of in vitro cultivation were detected within three populations. Results are discussed in relation to known facts.