Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 120-134).

Orientador: José Buratini Junior / Resumo: A comunicação entre o oócito e células somáticas adjacentes é essencial para a diferenciação do complexo cumulus-oócito (COC). Essa comunicação é proporcionada por processos citoplasmáticos trans-zonais (TZP) que penetram através da zona pelúcida e atingem a membrana do oócito, onde junções do tipo gap permitem transporte bidirecional de sinais elétricos e moléculas reguladoras. A maturação oócitária in vivo é controlada por gonadotrofinas e peptídeos intra-ovarianos e desencadeada pelo pico de LH. Este, induz a expressão dos fatores EGF-like que desencadeiam uma série de fatores que levam a fosforilação das conexinas, e com isso o fechamento de junções gap e consequentemente perda dos efeitos inibitórios do monofosfato cíclico de adenosina (AMPc) e monofosfato cíclico de guanosina (GMPc), proporcionando assim a retomada da meiose. O objetivo deste trabalho é avaliar os efeitos da inclusão de uma fase de pré maturação seguida da maturação in vitro sobre a progressão da meiose, funcionalidade das junções gap e produção embrionária. A pré maturação foi empregada utilizando componentes de ordem fisiológica encontradas no ambiente folicular, e esta, foi capaz de retardar a quebra da vesícula germinativa (VGBD), enquanto favoreceu a comunicação entre oócito e células do cumulus, o que acreditamos ser beneficial para o desenvolvimento oocitário in vitro. No entanto, quando seguida da maturação in vitro também utilizando componentes fisiológicos, foi capaz de prod... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The maturation of the cumulus oocyte complex (COC) is triggered by the ovulatory LH surge in vivo, and precociously induced when the COC is removed from the follicle. Natriuretic peptide type C (NPPC) plays a central role in maintaining meiotic arrest in vivo and delays GVBD while increasing oocytecumulus communication during IVM in cattle. In this study we first tested the hypothesis that follicular steroids (estradiol, progesterone and androstenedione) combined with FSH at approximately physiological levels cooperate with NPPC to delay GVBD and to maintain oocyte-cumulus communication. Then, we assessed the effects of follicular steroids, NPPC and the combination of both in a pre-IVM culture followed by IVM induced with AREG on embryo production. Follicular steroids combined with NPPC was most effective in increasing the proportion of GV oocytes in Bos indicus, although follicular steroids did not significantly enhance the effect of NPPC in Bos Taurus. . Moreover, follicular steroids enhanced the ability of NPPC to increase the proportion of COCs with opened GAP junctions. The addition of follicular steroids and/or NPPC to a pre- IVM culture did not alter blastocyst production after IVF. In conclusion, the combination of follicular steroids with NPPC can alter GVBD dynamics and cell communication favorably to oocyte developmental competence, but when used in a pre-IVM culture followed by induction of COC maturation with AREG and IVF, it did not increase e... (Complete abstract click electronic access below) / Mestre

Master of Science / Department of Biochemistry / Dolores J. Takemoto / The vertebrate lens relies on gap junction-mediated intercellular communication to maintain cellular homeostasis and lenticular transparency. Differentiation of cuboidal lens epithelial cells into cortical fiber cells involves the degradation of endogenous gap junction protein, connexin 43 (Cx43) and the up-regulation of connexin 46 (Cx46). Cx46 may also be involved in the hypoxia response in other tissues; a function that can possibly be attributed to unique phosphorylation sites at the cytoplasmic C-terminus. In this study, we have developed a mammalian (rabbit) lens epithelial cell (RLEC) culture model that overexpresses Cx46 to ascertain the role of Cx46 in differentiation and oxidative stress response. The cell line N/N1003A was stably transfected with a GFPCx46 plasmid construct, and analyzed for differentiation markers including endogenous gap junction protein isoforms (Cx43 and Cx50). Western blot analysis and visual observation determined that the stable overexpression of Cx46 (sCx46OE) induced the degradation of Cx43 and elicited morphological changes indicative of fiber cell elongation. Total RNA from RLEC culture was isolated and analyzed for mRNA levels using RT-PCR. Comparable levels of Cx43 transcript were present in wild type, transient Cx46OE (tCx46OE), and sCx46OE which suggests a post-transcriptional regulation of Cx43 degradation. Treatment of sCx46OE with proteasome inhibitors restored Cx43 protein levels, and scanning confocal microscopy supported our hypothesis that Cx43 is degraded in differentiating lens cells by way of a ubiquitinmediated proteasomal pathway. It is our conclusion that Cx46 has application in hypoxic conditioning and differentiation in addition to its conventional role as a gap junction protein.

Striatal oscillatory activity associated with movement, reward, and decision-making is observed in several interacting frequency bands. Rodent striatal local field potential recordings show dopamine- and reward-dependent transitions between a 'spontaneous' state involving beta (15-30 Hz) and low gamma (40-60 Hz) and a 'dopaminergic' state involving theta (4-8 Hz) and high gamma (60-100 Hz) activity. The mechanisms underlying these rhythmic dynamics and their functional consequences are not well understood. In this thesis, I construct a biophysical model of striatal microcircuits that comprehensively describes the generation and interaction of these rhythms as well as their modulation by dopamine and rhythmic inputs, and test its predictions using human electroencephalography (EEG) data. Chapter 1 describes the striatal model and its dopaminergic modulation. Building on previous work suggesting striatal projection neuron (SPN) networks can generate beta oscillations, I construct a model network of striatal fast-spiking interneurons (FSIs) capable of generating delta/theta (2-6 Hz) and gamma rhythms. This FSI network produces low gamma oscillations under low (simulated) dopaminergic tone, and high gamma activity nested within a delta/theta oscillation under high dopaminergic tone. In a combined model under high dopaminergic tone SPN network beta oscillations are interrupted by delta/theta-periodic bursts of gamma-frequency FSI inhibition. This high dopamine-induced periodic inhibition may enable switching between beta-rhythmic SPN cell assemblies representing motor programs, suggesting that dopamine facilitates movement in part by allowing for rapid, periodic changes in motor program execution. Chapter 2 describes the model's response to square-wave periodic cortical inputs. Comparing models with and without FSIs reveals that the FSI network: (i) prevents the SPN network's generation of phase-locked beta oscillations in response to beta's harmonic frequencies, ensuring fidelity of transmission of cortical beta rhythms; and (ii) limits or entrains SPN activity in response to certain gamma frequency inputs. Chapter 3 describes an analysis of phase-amplitude coupling at cortical electrodes in human EEG data during a reward task. The alternating rhythms predicted by the model appear in response to positive feedback. While the origins of these rhythms remain unclear, if they represent striatal signals, they provide a direct link between human behavior and striatal cellular function.

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Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.

Calcium, a pivotal ion in various human signaling pathways, holds significant relevance in cancer research and treatment. Investigating the transmission of spontaneous calcium signals between cells and understanding how they are influenced by different treatments is therefore an interesting area of study. This master's thesis presents a newly developed tool designed for analyzing intracellular calcium concentration [Ca2+] fluctuations. The tool encompasses a cell segmentation model, tracking algorithms, nearest neighbor identification, mean intensity measurement, and calculations of dominant frequency of [Ca2+] oscillations and signal correlation among neighboring cells. The tool was applied to fluorescence microscopy images of Madin-Darby Canine Kidney II cells expressing GCaMP6m and either treated with the cardiac glycoside ouabain, a combination of ouabain and the gap junction blocker heptanol, or left untreated for comparison. Additionally, an exploration into the impact of connexin 43 (Cx43) transfection on [Ca2+] fluctuations was undertaken. The transfection rate for Cx43 did not reach a sufficient level, impeding a thorough analysis of its influence on [Ca2+] fluctuations. Furthermore, the investigation into dominant frequencies and signal correlation did not yield conclusive findings regarding the effects of ouabain and heptanol. Nevertheless, a foundational tool for analysis has been developed, with the potential for expansion to analyze more aspects calcium signaling and application to additional cell types and treatments. / Kalcium, en central jon i olika signalvägar hos människan, spelar en viktig roll inom cancerforskning och -behandling. Att undersöka överföringen av spontana kalciumsignaler mellan celler och förstå hur dessa påverkas av olika behandlingar är därav ett intressant studieområde. I denna masteruppsats presenteras ett nyutvecklat verktyg utformat för att analysera variationer i intracellulär kalciumkoncentration [Ca2+] . Verktyget omfattar en cellsegmenteringsmodell, spårningsalgoritmer, identifiering av närmaste grannar, medelintensitetsmätning, beräkningar av dominant frekvens för oscillationer i [Ca2+] samt korrelation i dessa variationer mellan närliggande celler. Verktyget tillämpades på fluorescensmikroskopibilder av Madin-Darby Canine Kidney II-celler som uttryckte GCaMP6m och antingen hade behandlats med hjärtglykosiden ouabain, en kombination av ouabain och gap junction-blockeraren heptanol eller lämnats obehandlade för jämförelse. Dessutom undersöktes hur variationerna i [Ca2+] påverkades om cellerna transfekterades med konnexin 43 (Cx43). Andelen celler per prov som genomgick lyckad transfektion av Cx43 nådde aldrig upp till önskade nivåer, vilket förhindrade närmare analys av påverkan på [Ca2+]. Undersökningen av dominanta frekvenser och korrelation i signaler gav inte heller några entydiga svar på hur ouabain och heptanol påverkar [Ca2+]. Likväl har ett grundläggande verktyg för analys utvecklas, som har potential att utvidgas till analys av fler aspekter av kalciumsignalering och tillämpning på fler celltyper och behandlingar.

Die vorliegende Dissertation befasst sich mit Entstehungsmechanismen kardialer Arrhythmien auf der Grundlage pathologisch veränderten Myokards. Es wurde eine systematische Analyse der elektrophysiologischen Veränderungen, die als Folge von Gap-Junction- Lateralisierung und zellulärer Hypertrophie auftreten, durchgeführt. Die Analyse beruht auf einem mathematischen Computermodell, das zur Simulation der Aktionspotentialausbreitung innerhalb einer Einzelzellschicht humaner ventrikulärer Kardiomyozyten entwickelt wurde. Ausgehend von bestehenden Einzelzellmodellen wurde ein räumlich und zeitlich hoch aufgelöstes Multizellmodell generiert und in der Programmiersprache Object Pascal implementiert. Nach Validierung des Modells wurde es zur gezielten, an experimentellen Daten orientierten Manipulation geometrischer Eigenschaften der Zellen (Länge, Durchmesser) und des Zellverbandes (Anordnung der Zellen untereinander) sowie der Gap-Junction-Verteilung genutzt. Die Analyse der elektrophysiologischen Effekte im Vergleich zur Kontrolle fand sowohl unter Normalbedingungen als auch unter Bedingungen, die pathologischen Veränderungen entsprechen (Entkopplung der Gap-Junctions, verringerte Aktivität des schnellen Natriumkanals, erhöhte Inhomogenität), statt. Es zeigte sich, dass ein größerer Zelldurchmesser bzw. erhöhte laterale Gap-Junction-Leitfähigkeit (Simulation von kardialer Hypertrophie bzw. Connexin- Lateralisierung) die Entstehungswahrscheinlichkeit eines unidirektionalen Leitungsblocks erhöhte. Die Erregungsausbreitungsgeschwindigkeit in hypertrophierten Zellen war zudem weniger stabil als in normalen Zellen. Beide Effekte gehören zu den Hauptursachen der Entstehung und Aufrechterhaltung ventrikulärer Arrhythmien. Die Ergebnisse der Arbeit erklären somit Ursachen des erhöhten Arrhythmierisikos in pathologisch veränderten und hypertrophierten Herzen und liefern eine theoretische Grundlage für zukünftige Studien.