<p>Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.</p> / Dissertation

Reflex effects evoked by graded electrical stimulation of the posterior articular nerves (PAN) of the ipsi- and contralateral knee joints were investigated using both micro-electrode recordings from 7 - motoneurones and recordings from single muscle muscle spindle afferents. Spindle afferent responses were also recorded using natural stimulation of different types of receptors, to elucidate if the articular reflexes onto the y -motoneurones were potent enough to significantly alter the muscle spindle afferent activity. Stretches of the ipsilateral posterior (PCL) and anterior (ACL) cruciate ligaments, pressure on the ipsi- and contralateral knee and ankle joint capsules, and passive flexion/extension movements of the joints in the contralateral hind limb were performed. The occurrance of different sensory endings in the ACL and PCL was examined using gold chloride staining for neuronal elements. All experiments were performed on chloralose anaesthetized cats. More than 90% of the static and dynamic y -motoneurones were responsive to electrical stimulation of the PAN. Most 7-cells responded to low intensity electrical stimulation. Excitatoiy reflex effects predominated on both static and dynamic posterior biceps-semitendinosus (PBSt) 7 -cells, while excitatory and inhibitory effects occurred with an about equal frequency on triceps-plantaris (GS) 7-cells. The fastest segmental route for excitatory PAN effects on hind limb 7-motoneurones seems to be di- or trisynaptic, while the path for inhibitory effects seems to be at least one synaps longer. Physiological stimulations of ipsi- and contralateral joint capsules and of ipsilateral cruciate ligaments were all found to evoke frequent and potent changes in spindle afferent responses from the GS and PBSt muscles. It was shown that these effects were due to reflexes onto dynamic and static fusimotor neurones caused by physiological activation of articular sensory endings. Both ipsi- and contralateral joint receptor stimulation evoked excitatory as well as inhibitory fusimotor effects. The highest responsiveness was found during stimula­tion of the cruciate ligaments, i.e. 58% for GS and 47% for PBSt primary spindle afferents to PCL stimula­tion, and 73% for GS and 55% for PBSt primary spindle afferents to ACL stimulation. Significant altera­tions in spindle afferent activity was encountered at very low traction forces applied to the cruciate ligaments (5-10 N). The low thresholds, the tonic character of the stimuli, and the fact that different types of sensory endings were demonstrated in the cruciate ligaments (i.e. Ruffini endings, Pacinian corpuscles, Golgi ten­don organ like endings and free nerve endings), indicate that the fusimotor effects observed were caused by activation of slowly adapting mechanoreceptors, most likely Ruffini endings and/or Golgi tendon organ like endings. The potent reflex effects on the muscle spindle afferents elicited by increased tension in the cruciate ligaments indicate that these ligaments may play a more important sensory role that hitherto believed, and it is suggested that they may be important in the regulation of the stiffness of muscles around the knee joint, and thereby for the joint stability. The possible clinical relevance and the mechanisms by which joint receptor afferents, via adjustment of the muscle stiffness, may control joint stability are discussed. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1989, härtill 7 uppsatser.</p> / digitalisering@umu

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 163-179).

Universiẗat, Diss., 2003--Düsseldorf.

Το σύμπλεγμα-Κ και η άτρακτος είναι από τα χαρακτηριστικότερα ηλεκτροεγκεφαλογραφικά στοιχεία του δευτέρου NREM σταδίου του ύπνου στον άνθρωπο. Η παρούσα μελέτη διεξήχθη με σκοπό να διερευνήσει πιθανές σχέσεις μεταξύ των φαινομένων αυτών που εμφανίζονται κατά το δεύτερο στάδιο του NREM ύπνου, με ταυτόχρονη φιλοδοξία να απαντήσει σε ερωτήματα σχετικά με τον λειτουργικό ρόλο αυτών. Δέκα υγιή υποκείμενα έλαβαν μέρος στην μελέτη, κατά την οποία ελήφθη το ολονύχτιο ηλεκτροεγκεφαλογράφημα του ύπνου τους. Τα φαινόμενα ενδιεφέροντος αναγνωρίστηκαν, επιλέχθησαν και σημειώθηκαν προκειμένου να υποβληθούν σε συμβαντο-σχετιζόμενη ανάλυση, η οποία ενισχύθηκε από την εφαρμογή ανάλυσης χρόνου-συχνοτήτων καθώς και διδιάστατης τοπογραφικής απεικόνισης στον χώρο των ηλεκτροδίων. Η παρούσα μελέτη οδήγησε στα παρακάτω ευρήματα σχετικά με τα κύρια ηλεκτροεγκεφαλογραφικά στοιχεία του δευτέρου NREM σταδίου του ανθρώπινου ύπνου. Ταχείες άτρακτοι στην πορεία των οποίων τυγχάνει να εμφανιστεί σύμπλεγμα-Κ διακόπτουν την ταλάντωσή τους. Άτρακτοι οι οποίες με μεγάλη πιθανότητα εμφανίζονται μετά την πάροδο του συμπλέγματος-Κ έχουν υψηλότερη συχνότητα ταλάντωσης τόσο από εκείνες που διεκόπησαν όσο και από τις σποραδικές, μη-συσχετισμένες με σύμπλεγμα-Κ, ατράκτους. Η εν λόγω αύξηση στην συχνότητα τείνει προς μια μέγιστη συχνότητα ταλάντωσης και δεν εξαρτάται από κάποιο από τα ηλεκτροεγκεφαλογραφικά καταγραφόμενα χαρακτηριστικά του συμπλέγματος-Κ. Αντίστοιχα, και η διακοπή της ταλάντωσης των ατράκτων πρό του συμπλέγματος-Κ δεν μπορεί με βεβαιότητα να αποδωθεί στο σύμπλεγμα-Κ. Εντός του συμπλέγματος-Κ εμφανίζεται με μεγάλη πιθανότητα ρυθμική δραστηριότητα στο άνω όριο της θήτα ζώνης συχνοτήτων. Η δραστηριότητα αυτή είναι ανεξάρτητη του συμπλέγματος-Κ, καθώς εμφανίζει κατά πλειονότητα προσθιο-οπίσθιο προφίλ διάδοσης όταν το αργό κύμα του συμπλέγματος-Κ έχει μετωπιαία εντόπιση. Ο υψηλός θήτα ρυθμός αυτός, όσο περισσότερες ταλαντώσεις επιτυγχάνει εντός του συμπλέγματος-Κ τόσο περισσότερο τείνει να εισέλθει συχνοτικά στην άλφα ζώνη συχνοτήτων, από την οποία κατά πλειονότητα επανέρχεται στα αρχικά συχνοτικά επίπεδα κατά την τελευταία ταλάντωση˙ μια φασματική επαναφορά προτύπου πέδησης, την οποία ελλείψει άλλων οπτικά αξιολογήσιμων στοιχείων αποδίδουμε στην ύστερη δράση του συμπλέγματος-Κ. Η μελέτη αυτή έδειξε: α) ότι το σύμπλεγμα-Κ και οι άτρακτοι αποτελούν υπναγωγικά στοιχεία του φλοιού και του θαλάμου αντίστοιχα, τα οποία αντικατοπτρίζουν ανεξάρτητες αλλά συνεργές διαδικασίες που σκοπό έχουν να διατηρήσουν την συνέχεια του ύπνου, και β) ότι οι αλληλεπιδράσεις του συμπλέγματος-Κ με τους περί αυτό ρυθμούς υποδεικνύουν την ύπαρξη μιας ακόμη πιο βραχύχρονης δυναμικής διεργασίας στην μικρο-αρχιτεκτονική του ύπνου. Παράλληλα η μελέτη οδήγησε στην ανάπτυξη ενός νέου εργαλείου ταυτόχρονης απεικόνισης της μακρο-αρχιτεκτονικής και της μικρο-αρχιτεκτονικής του ύπνου: του υπνοφασματογραφήματος, του διαγράμματος χρόνου-συχνοτήτων του ολονύχτιου ηλεκτροεγκεφαλογραφήματος του ύπνου. / The K-complex and the sleep spindle are the most distinctive electroencephalographic features of the the second stage of NREM human sleep. This study was performed in order to investigate the potential relations between these phenomena that make their appearance in the second stage of NREM sleep, at the same time aiming in answering questions regarding their physiological role. Ten healthy individuals participated in this study, during which the whole-night sleep electroencephalogram was derived. The features of interest were identified, selected and marked in order to undergo event-related analysis, empowered by time-frequency analysis and 2-dimentional topography in electrode space. This study led to the following conclusions regarding the main electroencephalographic features of the second stage of NREM human sleep. Fast sleep spindles, during the course of which a K-complex happens to appear, interrupt their oscillation. The spindles that appear with high probability after the K-complex have higher frequency of oscillation from both the interrupted ones as well as form the sporadic, non-cprrelated to the K-complex, spindles. This increment in frequency tends towards a maximum oscillation frequency and is not dependent in any of the electroencephalographically recorded features of the K-complex. Respectively, the interruption of the oscillation of the pre-K-complex spindles cannot be credited to the K-complex with certainty. During the course of the K-complex a rhythmic activity of the upper theta band appears with high probability. This activity is independent from the K-complex as, in majority, it exhibits a antero-posterior profile of displacement when the slow wave of the K-complex has a frontal localization. This high-theta rhythm, the more oscillation peaks it achieves during the K-complex, the more it tends to enter the alpha band, from which, in majority, it returns to the initial frequency levels during the last oscillation; a spectral breaking profile that, in the absence of other visually evaluated elements, we credit to the late action of the K-complex. This study showed that: a) the K-complex and the sleep spindle are sleep-promoting elements of the cortex and the thalamus, respectively, that represent independent but cooperative processes that aim in preserving the continuity of sleep, and b) that the interactions of the K-complex with the rhythms around it reveal the existence of an even briefer dynamic process of the micro-architecture of sleep. At the same time, this study led to the development of a novel tool for concurrent visualization of the macro-architecture and the micro-architecture of sleep: the hypnospectrogram, the time-frequency plot of the whole-night sleep electroencephalogram.

<p>The ultimate goal of any living cell is to pass on a complete, unaltered copy of its DNA to its daughter cell. The DNA damage response (DDR) and spindle checkpoint are two essential signaling pathways that make it possible for a cell to achieve this goal. The DDR protects genetic integrity by sensing errors in the DNA sequence and activating signaling pathways to arrest the cell cycle and repair the DNA. The spindle checkpoint protects chromosomal integrity by preventing the separation of chromosomes during mitosis until all chromosomes are correctly attached to the mitotic spindle. Proper regulation of both the DDR and the spindle checkpoint is critical for cell survival. In this dissertation I will describe our discovery of novel regulatory mechanisms involved in each of these signaling networks.</p><p>In the first research chapter of this dissertation, we describe our findings concerning how the DDR regulates cyclin F levels. Cyclin F is an F-box protein that associates with the SCF E3 ubiquitin ligase complex to target proteins for degradation. In response to DNA damage, cyclin F levels are downregulated to facilitate increased dNTP production for efficient DNA repair, but the molecular mechanisms regulating this downregulation of cyclin F are largely unknown. We discovered that cyclin F downregulation by the DDR is the combined result of increased protein degradation and decreased mRNA expression. At the level of protein regulation, cyclin F is targeted for proteasomal degradation by the SCF complex. Interestingly, we found that the half-life of cyclin F protein is significantly increased in cells treated with the phosphatase inhibitor calyculin A, which caused cyclin F to be hyper-phosphorylated. Calyculin A also partially prevented cyclin F downregulation following DNA damage. This result suggests that cyclin F phosphorylation stabilizes the protein, and dephosphorylation of cyclin F may be required for its degradation in both unperturbed and DNA damaged cells. We also found that cyclin F downregulation is dependent on the Chk1 kinase, which is predominately activated by the ATR kinase. In examining the mechanism by which Chk1 promotes cyclin F downregulation, we determined that Chk1 represses cyclin F transcription. Lastly, we investigated the role of cyclin F in cell cycle regulation and discovered that both increased and decreased cyclin F expression delay mitotic entry, indicating that an optimal level of cyclin F expression is critical for proper cell cycle progression.</p><p>The second research chapter of this dissertation details our discovery of the requirement for phosphatase activity to inhibit the APC/C E3 ubiquitin ligase during the spindle checkpoint. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C until the chromosomes are properly aligned and attached to the mitotic spindle at metaphase. Once all the chromosomes are properly attached to the spindle, the MCC dissociates, and the APC/C targets cyclin B and securin for degradation so that the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. In our studies, we sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined their effect on the MCC and APC/C activation. Using this approach we found that two phosphatase inhibitors, calyculin A and okadaic acid (1 µM), caused MCC dissociation and APC/C activation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.</p> / Dissertation