Dissertationen zum Thema „CCDC80“

The ccdc80 and ccdc80-like1 (ccdc80-l1) genes were isolated in zebrafish in silico, on the basis of their high aminoacidic sequence identity with the human CCDC80 protein (coiled-coil domain containing 80). In human, CCDC80 is involved in several carcinomas, and during recent years it has been proposed as an onco-suppressor gene, increasing the interest in the comprehension of its functions. During my Ph.D., I have been studying the expression patterns and the functions of its zebrafish homologs, in order to gain insights into the processes and the molecular mechanisms in which they are involved. I took advantage of zebrafish as a model system allowing the application of common genetic and biological experimental assays such as PCR, whole mount in situ hybridization and microinjection technique. I investigated ccdc80 and ccdc80-l1 functions during zebrafish embryonic development, finding that they show very different expression patterns and roles. ccdc80 is expressed in the notochord during somitogenesis up to 48 hour post fertilization (hpf). At this stage, it is expressed also in the heart. Instead, at the same developmental stages, ccdc80-l1 is expressed in cranial ganglia, adaxial cells, muscle pioneers and dorsal dermis. These patterns are suggestive of different roles, in fact, the genes are involved in distinct developmental processes, such as somitogenesis and axonal pathfinding, respectively. Functional analysis were obtained performing loss- and gain-of-function experiments. The results clearly demonstrated that manipulation of Ccdc80 protein levels during embryonic development, both increasing and decreasing them, leads to a severe impairment of somites, metameric structures from which several tissues derive, such as muscle. Nevertheless, this phenotype seemed to be recovered at 24 hpf, since at this stage somites no longer showed the same morphological alterations observed previously. By converse, notwithstanding its expression in muscular tissues, loss-of-ccdc80-l1 does not impair somites formation, leading instead to motoneurons axonal migrations defects, capable to affect embryonic motility. Given the external development of the zebrafish embryo, motility is required very soon for prey and escaping predators, and muscle and motoneurons are equally required for a proper motor behavior. Communication and interactions between these two tissues are fundamental for proper muscles innervation and transmission of nervous inputs to be translated in muscular contractions. ccdc80 and ccdc80-l1 may derive from an ancestor gene involved in these processes and, during evolution, may have developed different functions, but still bound to a key process for embryonic viability in the external environment.

Background: The gene Coiled Coil Domain Containing 80 (Ccdc80) is widely expressed in normal human tissues, at particularly high levels in heart, skeletal muscle and adipose tissue. Its role is well defined in tumor suppression, axon path finding, glucose homeostasis, bone marrow stromal cells and adipocyte differentiation. Moreover, it plays a role in embryonic development of lens and muscle, but no clear-cut data are available about the role of Ccdc80 in heart development and in heart disease. Aims: To define if Ccdc80 is expressed during embryonic development of zebrafish heart and if its functional block causes alterations of heart structure or contractility; to define the expression pattern of Ccdc80 protein in normal heart vs cardiomyopathies in humans (samples taken from patients with dilated cardiomyopathy – DCM) and rodents (samples taken from right ventricle heart failure induced by monocrotaline – MCT). Results: In zebrafish Ccdc80 is widely expressed in the forming heart, during all the developmental stages; Ccdc80-morphants show defects in the developing heart, with impaired cardiac looping, atrium enlargement, blood stasis and peripheral congestion. These phenotypical alterations, similar to heart failure, are due to a disorder of the late phase of cardiac development, after myocyte differentiation. In normal human and rats, Ccdc80 mRNA, analized by Northern blot technique, showed a higher expression in atria compared to ventricles, while the Ccdc80 protein (108 Kd), analized by Western blot, showed similar expression levels in atria and ventricles. Ccdc80 protein showed different expression patterns, in atria and ventricles with a cytoplasmic localization and co-localization with sarcomeric proteins at immunofluorescence analysis. In pathological samples (DCM and MCT rats) Ccd80 protein showed evident overexpression and different isoforms, related to protein phosphorylation and secreted protein isoform, suggesting a different feature in pathological conditions compared to normal. Conclusions: Our results demonstrated that Ccdc80 has an indispensable role for correct heart development. In complete developed heart, Ccdc80 showed an adaptive function to stress conditions, such as pressure overload, and in cardiomyopathies showed increased expression and different isoforms
Introduzione: Il gene Coiled Coil Domain Containing 80 (Ccdc80) è ampiamente espresso in tessuti umani normali, a livelli particolarmente elevati nel cuore, muscolo scheletrico e tessuto adiposo. E’ ben definito il suo ruolo come oncosoppressore, nella innervazione degli assoni, nella omeostasi glicemica, e nel differenziamento delle cellule stromali del midollo osseo e degli adipociti. Inoltre, svolge un ruolo nello sviluppo embrionale muscolare e della lente del cristallino, ma non sono disponibili dati sul ruolo di Ccdc80 nello sviluppo del cuore e nelle malattie cardiache. Obiettivi : Definire se Ccdc80 è espresso durante lo sviluppo embrionale del cuore zebrafish e se il suo blocco funzionale provoca alterazioni della struttura o della contrattilità; definire il pattern di espressione della proteina Ccdc80 nel cuore normale vs cardiomiopatie nell'uomo (campioni prelevati da pazienti con cardiomiopatia dilatativa - DCM ) e roditori (campioni prelevati da ratti con insufficienza ventricolare destra indotta da monocrotalina - MCT). Risultati: In zebrafish Ccdc80 è ampiamente espresso nel cuore in formazione, durante tutte le fasi di sviluppo; i morfanti per Ccdc80 mostrano difetti nello sviluppo cardiaco, con alterato looping, dilatazione atriale, stasi ematica e congestione periferica. Queste alterazioni fenotipiche, simili ad uno scompenso cardiaco, sono dovuti ad un disturbo della fase tardiva dello sviluppo cardiaco, dopo che la differenziazione dei miociti è già stata completata. Nei campioni normali di uomo e ratto, l’RNA messaggero di Ccdc80, analizzato con tecnica di Northern blot, è risultato maggiormente espresso negli atri rispetto ai ventricoli, il mentre l’espressione della proteina Ccdc80 (108 Kd), analizzata con tecnica Western blot, è risultata simile negli atri e nei ventricoli. La proteina Ccdc80 ha mostrato pattern di espressione diversi in atri e ventricoli, con localizzazione citoplasmatica e chiara co-localizzazione con le proteine sarcomeriche all’immunofluorescenza. Nei campioni patologici (DCM ed MCT ratti) la proteina Ccd80 ha mostrato una netta iper-espressione sia negli atri che nei ventricoli, e la presenza di diverse isoforme, suggerendo diverse funzioni in condizioni patologiche rispetto al normale . Conclusioni: I nostri risultati hanno dimostrato che Ccdc80 ha un ruolo indispensabile nello sviluppo cardiaco. Nel cuore adulto, Ccdc80 si manifesta con diverse isoforme e maggiore espressione, rivestendo una funzione adattativa in condizioni di stress, come il sovraccarico di pressione, e nelle cardiomiopatie

Intracellular signaling requires rapid and efficient changes in the intracellular concentration of messengers through time. One of most important second messenger is Calcium. Mitochondria are important component in intracellular signaling and Ca2+ is one of the key regulators of organelle physiology. Its uptake inside the organelle is driven by the potential across the inner mitochondrial membrane, through a selective ion channel, called Mitochondrial Calcium Uniporter (MCU) (De Stefani, D., et al., Nature (2011); Baughman, J.M., et al., Nature (2011)). Ca2+ plays a pleiotropic role into mitochondria, ranging from the regulation of ATP production, to the shaping of cytoplasmic Ca2+ waves and activation of apoptosis (Rizzuto, R., et al., Nat Rev Mol Cell Biol (2012)). MCU is the core element of the so-called “MCU complex”. Several pathological conditions are directly or indirectly linked to mitochondrial dysfunction, and the molecular characterization of the MCU complex, that is responsible for the highly selective transport of Ca2+ across the inner mitochondrial membrane, gives the opportunity to modulate mitochondrial function by modifying MCU channel activity. The mechanism by which Ca2+ is transported into mitochondria has been unclear for a long time and several proteins have been identified in the past years. We now know that the MCU complex is formed by MCU, the selective channel that allows Ca2+ entry into the matrix, its dominant negative isoform MCUb, the Essential MCU REgulator (EMRE) and two channel modulators, Mitochondrial Calcium Uptake 1 and Mitochondrial Calcium Uptake 2 (MICU1 and MICU2) (De Stefani, D. and Rizzuto,R., Biochem Biophys Res Commun (2014)). Recently, another regulator was discovered and named MCU Regulator 1 (MCUR1). MCUR1 down regulation causes a decrease in agonists-induced mitochondrial calcium transients; moreover, MCUR1 is able to bind MCU but not MICU1, thus suggesting that MCU may exists in two different complexes, one with MCUR1 and another one with MICU1 (Mallilankaraman, K., et al., Nat Cell Biol (2012)) . We found that MCUR1 (formerly known as CCDC90A) has also an isoform, named CCDC90B, whose function is still totally unknown. This work is thus focused on the role of these two proteins in the regulation of the MCU complex activity. We found that both MCUR1 and CCDC90B are integral proteins of the inner mitochondrial membrane, they are broadly expressed among tissues, and CCDC90B mRNA is generally present at higher levels than MCUR1. From the functional point of view, silencing of MCUR1 or CCDC90B leads to a decrease of mitochondrial calcium influx. However, we could detect only a marginal functional interaction between these two proteins and the other components of the MCU complex. Accordingly, the down regulation of both these proteins decreases mitochondrial membrane potential. Overall our data suggest that MCUR1 and its isoform CCDC90B have an indirect effect on mitochondrial calcium uptake, due to a mechanism that we are currently addressing and could depend on the assembly of ETC complexes (Paupe, V., et al., Cell Metabolism (2015)).
I meccanismi di trasduzione di segnali intracellulari richiedono cambiamenti rapidi ed efficienti della concentrazione di molecole segnale nel tempo. Uno dei più importanti secondi messaggeri è il Calcio (Ca2+). I mitocondri sono fondamentali nella segnalazione intracellulare e il Ca2+ è a sua volta un elemento chiave della fisiologia di questi organelli. L’accumulo di Ca2+ all’interno dei mitocondri è guidato dal potenziale di membrana (Ψm), attraverso un canale ionico selettivo chiamato Mitochodrial Calcium Uniporter (MCU) (De Stefani, D., et al., Nature (2011); Baughman, J.M., et al., Nature (2011)). Il Ca2+ svolge un ruolo pleiotropico nei mitocondri, che va dalla regolamentazione della produzione di ATP, al controllo delle onde di Ca2+ citoplasmatico fino all’attivazione dell’apoptosi cellulare (Rizzuto, R., et al., Nat Rev Mol Cell Biol (2012)). MCU è l’elemento principale dell’omonimo complesso denominato “complesso MCU”. Diverse condizioni patologiche sono direttamente o indirettamente legate a disfunzioni delle funzioni mitocondriali. La caratterizzazione molecolare del complesso di MCU, responsabile del trasporto selettivo degli ioni Ca2+ attraverso la membrana mitocondriale interna (IMM), offre la possibilità di modulare l’attività del canale MCU, fondamentale per la funzione signaling mitocondriale. Il meccanismo con il quale lo ione Ca2+ è trasportato all’interno dei mitocondri è tuttavia rimasto un mistero per molto tempo. Negli ultimi anni di ricerca diverse proteine sono state identificate come parte del complesso di MCU. Attualmente i protagonisti nell’attività di uptake di Ca2+ nel mitocondrio sono: MCU, il canale selettivo che permette l’accesso esclusivo agli ioni Ca2+ nella matrice mitocondriale; la sua isoforma MCUb, che svolge un effetto dominante negativo sull’attività del canale; EMRE, un regolatore essenziale di MCU e infine i due modulatori del canale MICU1 e MICU2, rispettivamente acronimi di Mitochondrial Calcium Uptake 1 e 2 (De Stefani, D. e Rizzuto, R., Biochem Biophys Res Commun (2014)) . Recentemente è stato scoperto un nuovo regolatore, chiamato MCU Regulator 1 (MCUR1). È stato provato che la riduzione dell’espressione di MCUR1 causa una rilevante riduzione della concentrazione di Calcio nella matrice mitocondriale. È inoltre riportato che MCUR1 è fisicamente legato a MCU, ma non presenta alcuna interazione fisica con MICU1, suggerendo la possibile esistenza di due tipologie di complessi MCU: uno con MICU1 e un altro con MCUR1, ma non con entrambi simultaneamente (Mallilankaraman, K., et al., Nat Cell Biol (2012)). In questo lavoro di ricerca è stato scoperto che MCUR1, conosciuto anche come CCDC90A, possiede un’isoforma, nota col nome di CCDC90B, e sua funzione è attualmente ancora sconosciuta. Pertanto abbiamo focalizzato la nostra attenzione su queste due proteine e la loro possibile funzione nel complesso di MCU. Entrambe le proteine, CCDC90A e CCDC90B, risiedono nella membrana mitocondriale interna, e sono espresse in tutti i tessuti umani. In particolare, in molti di essi l’mRNA di CCDC90B è generalmente presente in quantità superiore rispetto a CCDC90A. Dal punto di vista funzionale, il silenziamento di entrambe le proteine causa una diminuzione del flusso di Ca2+ mitocondriale. Tuttavia, entrambe le proteine hanno dimostrato solo una minima interazione funzionale con gli altri componenti del complesso di MCU. Inoltre, il silenziamento di CCDC90A e CCDC90B causa una significativa depolarizzazione del potenziale di membrana mitocondriale. Complessivamente, i dati raccolti in questo lavoro suggeriscono che sia CCDC90A che la sua isoforma CCDC90B hanno un effetto indiretto sull’accumulo mitocondriale di Ca2+. Probabilmente, questo effetto è correlato all’azione che queste due proteine potrebbero svolgere nell’assemblaggio dei complessi della catena di trasporto degli elettroni (mETC) (Paupe, V., et al., Cell Metabolism (2015)).

El WAT se considera un tejido dinámico que secreta factores y dirige señales a otros tejidos, modulando a nivel sistémico el metabolismo de lípidos, la acción de la insulina, la homeostasis metabólica y la inmunidad. La acumulación grasa en VAT suele ir acompañada de una activación e infiltración de los ATMs, es responsable de una inflamación de bajo grado y de la liberación de factores promotores de insulinorresistencia. Las adipoquinas, son secretadas por los adipocitos y participan en el diálogo endocrino con otros tejidos. En la obesidad, el perfil de expresión de adipoquinas cambia en respuesta a la cantidad y la condición del órgano adiposo. En esta tesis se han estudiado dos potenciales adipoquinas: PTX3 y CCDC80. PTX3 aparece desregulada en la obesidad y el síndrome metabólico, sus niveles circulantes se correlacionaron inversamente con adiposidad, niveles de leptina, CRP e IL-6 en individuos sanos. Es este estudio demostró que los valores plasmáticos de PTX3 en una cohorte de 27 individuos delgados y 48 con sobrepeso correlacionaron negativamente con los niveles basales de triglicéridos y la secreción a insulina tras OGTT. En una segunda cohorte compuesta de 19 individuos delgados, 28 con sobrepeso y 15 obesos se observó una asociación inversa con el peso corporal y la relación cintura/cadera. En VAT se hallaron niveles de mRNA mayores en obesos que en delgados, correlacionando positivamente con los de IL-1β, y mayores en la fracción adipocitaria que en la estromovascular. En adipocitos SGBS cultivados, la expresión se incrementó en respuesta a IL-1β y TNFα pero no a IL-6 o insulina. PTX3 podría cumplir un papel en el control metabólico. Su expresión génica aumenta en VAT en la obesidad, pese a encontrarse unos menores niveles plasmáticos, y en respuesta a citoquinas proinflamatorias en adipocitos cultivados. CCDC80 es una proteína secretada por los adipocitos que regula la homeostasis energética en ratones con obesidad inducida por dieta. Su relación con la obesidad se desconocía. Nuestro estudio encontró un contenido en CCDC80 60% menor en VAT que en SAT en individuos delgados. Éste se vio incrementado en VAT de individuos obesos respecto a delgados. En una cohorte con distintos grados de obesidad, los niveles plasmáticos correlacionaron negativamente con la secreción de insulina y los niveles de glucosa tras OGTT, y positivamente con el recuento de neutrófilos sanguíneos y los niveles de MCP-1 circulantes. En otra cohorte de obesos mórbidos se encontraron asociaciones con el grado de esteatosis, los niveles circulantes de CRP y el espesor íntima-media carotídeo. Así, CCDC80 aparece sobreexpresada en VAT en obesidad y sus niveles se asocian con una mejora en la tolerancia a la glucosa pero también con marcadores inflamatorios, independientemente de la obesidad. En la obesidad mórbida, se asocian con un desajuste del perfil metabólico, incluyendo inflamación, hígado graso y enfermedad vascular. El paso limitante en la entrada y β-oxidación de FA de cadena larga es su transporte al interior celular y mitocondria mediante FAT o FATP1l y CPTs o CACT. FATP1 es capaz de incrementar la captación de ácidos grasos en músculo aunque su localización subcelular y mecanismo de acción, continúan siendo discutidos. Además, FATP1 podría dirigir el destino matebólico de los FAs captados. Nuestro estudio localizó FATP1 en fracciones de membrana externa e intermembrana mitocondriales en músculo esquelético de ratón, lo que podría determinar sus efectos metabólicos. La sobreexpresión de FATP1 incrementó la disponibilidad tanto de ácidos grasos sistémicos como triglicéridos intramusculares. No contribuyó a la desregulación metabólica inducida por la dieta grasa. Sin embargo, FATP1 produjo una hipercetonemia, probablemente secundaria al ahorro en la oxidación de cuerpos cetónicos resultante del incremento de la oxidación de los ácidos grasos muscular.
WAT is a dynamic tissue that secretes factors to modulate sistemic lipid metabolism, insulin action, metabolic homeostasis and immunity. Fat accumulation in VAT is often accompanied by activation and infiltration of ATMs, is responsible for a low-grade inflammation and of the release of factors promoting insulin resistance. Adipokines are secreted by adipocytes and participate in the endocrine dialogue with other tissues. In obesity, the adipokine expression profile changes in response to the amount and condition of adiposity. We have explored two potential adipokines: PTX3 and CCDC80. PTX3 seems to be deregulated in obesity and metabolic syndrome, its circulating levels are inversely correlated with adiposity, leptin, CRP and IL-6 levels in healthy individuals. We have observed that PTX3 gene expression increased in VAT in obesity, despite lower plasma levels, and in response to proinflammatory cytokines in cultured SGBS adipocytes. CCDC80 is secreted by adipocytes and regulates energy homeostasis in mice with diet-induced obesity, its relationship with human obesity was unknown. We showed that CCDC80 is overexpressed in VAT in obesity and its levels are associated with an improvement in glucose tolerance, but with inflammatory markers independently of obesity. In morbid obesity, its levels are associated with an imbalance in the metabolic profile, including inflammation, fatty liver and vascular disease. The limiting step in the uptake and β-oxidation of long-chain FAs is their transport into the cell and mitochondria using FAT or FATP1, and CPTs or CACT. FATP1 increases FAs uptake into muscle although its subcellular location and mechanism of action are unknown. Our study located FATP1 in outer mitochondrial membrane and intermembrane space fractions in mouse skeletal muscle. FATP1 overexpression enhanced disposal of both intramuscular triglycerides and systemic fatty acids. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids in muscle.

LncRNAs are increasingly being recognised as functionally important for regulation of biological processes. We have identified an lncRNA which we believe is integral in lineage commitment during haematopoiesis. Here, we report that CCDC26 lncRNA is a regulator of β-globin and c-MYC gene expression. Indeed, CCDC26 silencing in erythroleukemic K562 cells led to several gene expression changes. Upon further investigation of genes linked to erythropoiesis, we observed the upregulation of β-globin expression. Our results suggest that CCDC26 regulates expression of β-globin and other genes by modulating epigenetic changes which could be important in linage-commitment. We have results to suggest the expression of β-globin and c-MYC genes may be synergistic and promotes differentiation of K562. Finally, RNAseq data further supports upregulation of differentiation specific genes further underpinning our hypothesis that this lncRNA may play an important role in lineage commitment.

3-M syndrome (named after three authors who first described the condition) is an autosomal recessive condition characterised by pre- and post-natal growth impairment, facial dysmorphism and radiological features (slender long bones and tall vertebral bodies). It is caused by loss of function mutations in the Cullin 7 (CUL7) and Obscurin-like 1 (OBSL1) genes. CUL7 is a protein involved in ubiquitination (the process of targeted protein degradation) and OBSL1 is a putative cytoskeletal adaptor protein. The mechanisms through which loss of function mutations in OBSL1 or CUL7 lead to growth impairment is unclear but previous work suggests impaired placental function and altered insulin-like growth factor 1 (IGF-1) signaling as possibilities. The overall aim of this study was to elucidate the mechanisms underlying growth impairment in 3-M syndrome. Initially phenotypic data was collected on a cohort of patients and a genotype-phenotype comparison was undertaken. Skin fibroblast cell lines were derived from four patients with 3-M syndrome and used to study growth hormone (GH) and IGF-1 signal transduction, cell proliferation and apoptosis. Subsequently a hypothesis generating approach to identify novel mechanisms underlying 3-M growth impairment was undertaken in whole transcriptome and metabolomic studies. In addition an animal model using morpholino oligonucleotide mediated knock down of OBSL1 in Xenopus tropicalis was developed to study the effects on growth in a non placenting vertebrate to determine if the growth impairment seen in 3-M syndrome is independent of placental function. Cell proliferation was reduced in 3-M fibroblasts while apoptosis was not different from controls. No differences in GH signal transduction were identified but reduced activation of AKT following IGF-1 stimulation was identified in 3-M fibroblast cell lines. IGF2 was identified as the top downregulated probeset in 3-M fibroblasts compared to control in the whole genome transcriptome analysis. Metabolomic changes related to energy metabolism were identified in 3-M syndrome fibroblasts. Knock down of xtOBSL1 using two independent morpholinos resulted in growth impairment at embryonic stage 50, suggesting the growth impairment seen is at least in part independent of placental function. These studies suggest impaired placental function is not a key component of the growth impairment in 3-M syndrome. Impairment of IGF-1 signal transduction and IGF2 silencing are likely to contribute to the growth impairment in 3-M syndrome. The mechanisms relating to this IGF2 silencing require further studies.

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPARγ expression and consequently leading to a drastic decrease of the PPARγ target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Cilia and flagella are hair-like projections found on the surface of virtually every vertebrate cell. These microtubule-based organelles are historically known for their striking motility, a valuable tool for the manipulation of fluid environments. In addition, immotile (or 'primary') cilia play critical roles in cell signaling. More than ten human diseases have been linked to cilia function, with pleiotropic phenotypes including obesity, kidney and liver disease, skeletal abnormalities, situs defects, mental retardation, and sterility. In this dissertation, I first examine the function of Cep290, a putative master regulator of cilia biology, which is mutated in five human ciliopathies. I found that the zebrafish Cep290 protein was localized in a cell-type specific fashion to two distinct ciliary compartments: transition zones and centriolar satellites. Through morpholino knockdown, I demonstrated that Cep290 regulates the length of photoreceptor, Kupffer’s vesicle, and spinal canal cilia, while it was dispensable for normal cilia length in other tissues. Rescue of Cep290 associated cilia length defects by overexpression of cilia membrane proteins implicated Cep290 in cilia vesicle trafficking. Unexpectedly, I found that Cep290 deficiency in Kupffer’s vesicle and spinal canal resulted in cilia paralysis, accounting for left right asymmetry and hydrocephalus phenotypes, and identifying a novel function for Cep290 in dynein arm assembly. In the second chapter I identify and characterize three novel ciliopathy genes. We performed a small-scale morpholino screen to test the function of predicted cilia proteins. Three genes essential to cilia motility were identified: c21orf59, ccdc65, and c15orf26. Parallel studies in other systems revealed that C21orf59 was a component of the flagellar matrix required for the assembly of outer dynein arms, while Ccdc65 was part of the dynein regulatory complex, which regulates ciliary beat patterns. Importantly, we discovered that both C21ORF59 and CCDC65 were mutated in patients diagnosed with the human motile cilia disorder primary ciliary dyskinesia, identifying two novel human disease genes. Taken together, this work analyses multiple requirements for the assembly of motile and primary cilia and highlights the utility of the zebrafish system in investigations of cilia biology, particularly in the discovery and characterization of human disease genes.

Master of Science
Department of Biochemistry
Qize Wei
Proper regulation of mitosis and cytokinesis is fundamentally important for all living organisms. During anaphase, antiparallel microtubules are bundled between the separating chromosomes, forming the central spindle (also called the spindle midzone), and the myosin contractile ring is assembled at the equatorial cortex. Regulators of central spindle formation and myosin contractile ring assembly are mostly restricted to the interdigitated microtubules of central spindles and they can be collectively called midzone components. It is thought that characteristic microtubule configurations during mitosis and cytokinesis are dictated by the coordinated action of microtubule-stabilizing and -destabilizing factors. Although extensive investigations have focused on understanding the roles of microtubule-bundling/stabilizing factors in controlling central spindle formation, efforts have been lacking in aiming to understand how microtubule-destabilizing factors regulate the assembly of central spindles. This dissertation describes the role of a novel microtubule-destabilizing factor termed CCDC69 (coiled-coil domain-containing protein 69) in controlling the assembly of central spindles and the recruitment of midzone components. Endogenous CCDC69 was localized to the nucleus during interphase and to the central spindle during anaphase. Exogenous expression of CCDC69 in HeLa cells destabilized microtubules and disrupted the formation of bipolar mitotic spindles. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and interfered with the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and pololike kinase 1 (Plk1) at the central spindle. CCDC69 knockdown also decreased equatorial RhoA staining, indicating that CCDC69 deficiency can impair equatorial RhoA activation and ultimately lead to cytokinesis defects. Four coiled-coil domains were found in CCDC69 and the C terminal coiled-coil domain was required for interaction with aurora B. Disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Further, vitro kinase assay showed that Plk1 could phosphorylate CCDC69. Taken together, we propose that CCDC69 acts as a scaffold and a microtubule-destabilizing factor to control the recruitment of midzone components and the assembly of central spindles.

Cybersecurity is an ever-expanding field. In order to stay current, training, development, and constant learning are necessary. One of these training methods has historically been competitions. Cybersecurity competitions provide a method for competitors to experience firsthand cybersecurity concepts and situations. These experiences can help build interest in, and improve skills in, cybersecurity. While there are diverse types of cybersecurity competitions, most are run with on-premise hardware, often centralized at a specific location, and are usually limited in scope by available hardware. This research focuses on the possibility of running cybersecurity competitions, specifically CCDC style competitions, in a public cloud environment. A framework for running cybersecurity competitions in general was developed and is presented in this research. The framework exists to assist those who are considering moving their competition to the cloud. After the framework was completed, a CCDC style competition was developed and run entirely in a public cloud environment. This allowed for a test of the framework, as well as a comparison against traditional, on-premise hosting of a CCDC. The cloud-based CCDC created was significantly less expensive than running a comparable size competition in on-premise hardware. Performance problems—typically endemic in traditionally-hosted CCDCs—were virtually non-existent. Other benefits, as well as potential contraindications, are also discussed. Another CCDC style competition, this one originally built for on-premise hardware, was then ported to the same public cloud provider. This porting process helped to further evaluate and enrich the framework. The porting process was successful, and data was added to the framework.

Domestic animals have rich phenotypic diversity that can be explored to advance our understanding of the relationship between molecular genetics and phenotypic variation. Since the advent of second generation sequencing, it has become easier to identify structural variants and associate them with phenotypic outcomes. This thesis details studies on three such variants associated with monogenic traits. The first studies on Rose-comb in the chicken were published over a century ago, seminally describing Mendelian inheritance and epistatic interaction in animals. Homozygosity for the otherwise dominant Rose-comb allele was later associated with reduced rooster fertility. We show that a 7.38 Mb inversion is causal for Rose-comb, and that two alleles exist for Rose-comb, R1 and R2. A novel genomic context for the gene MNR2 is causative for the comb phenotype, and the bisection of the gene CCDC108 is associated with fertility issues. The recombined R2 allele has intact CCDC108, and normal fertility. The dominant phenotype Greying with Age in horses was previously associated with an intronic duplication in STX17. By utilising second generation sequencing we have examined the genomic region surrounding the duplication in detail, and excluded all other discovered variants as causative for Grey. Dun is the ancestral coat colour of equids, where the individual is mostly pale in colour, but carries intensely pigmented primitive markings, most notably a dorsal stripe. Dun is a dominant trait, and yet most domestic horses are non-dun in colour and intensely pigmented. We show that Dun colour is established by radially asymmetric expression of the transcription factor TBX3 in hair follicles. This results in a microscopic spotting phenotype on the level of the individual hair, giving the impression of pigment dilution. Non-dun colour is caused by two different alleles, non-dun1 and non-dun2, both of which disrupt the TBX3-mediated regulation of pigmentation. Non-dun1 is associated with a SNP variant 5 kb downstream of TBX3, and non-dun2 with a 1.6 kb deletion that overlaps the non-dun1 SNP. Homozygotes for non-dun2 show a more intensely pigmented appearance than horses with one or two non-dun1 alleles. We have also shown by genotyping of ancient DNA that non-dun1 predates domestication.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2016.
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Introdução. Evidências indicam um promissor efeito antitumoral das quinonas juglona, fenilaminonaftoquinona-7 (Q7) e fenilaminonaftoquinona-9 (Q9), especialmente quando associadas ao ascorbato. Objetivo. Dar continuidade aos estudos sobre o efeito antitumoral das naftoquinonas juglona, Q7 e Q9 em associação ao ascorbato de sódio, bem como avaliar a melhor associação para o tratamento do câncer a partir dos efeitos obtidos in vitro e in vivo. Metodologia. Foram analisados os efeitos das quinonas juglona, Q7 e Q9 associadas ou não ao ascorbato de sódio sobre o CT-DNA (Calf Thymus-DNA). Em células MCF-7 (carcinoma de mama) foram avaliados a viabilidade celular, os níveis de EROs intracelular, os danos ao DNA e a proliferação celular. Proteínas oriundas dos lisados de células MCF-7 foram utilizados para os ensaios de imunoeletroforese para verificar a integridade da proteína poli (ADP-ribose) polimerase (PARP), gama-H2AX (?H2AX) e pAkt. A atividade antitumoral in vivo das quinonas associadas ou não ao ascorbato de sódio foi inicialmente analisada através de parâmetros de sobrevida e crescimento tumoral de camundongos portadores do tumor ascítico de Ehrlich. Posteriormente, amostras do tumor foram coletadas e utilizadas para análise de marcadores de estresse oxidativo como: substâncias reativas ao ácido tiobarbitúrico (TBARS), proteína carbonilada, glutationa reduzida (GSH), superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa-S-transferase (GST). Ainda em amostras do tumor ascítico de Ehrlich foram avaliados danos ao DNA, parada do ciclo celular, influência na captação de glicose e tipo de morte celular induzida pelos tratamentos. As mesmas amostras foram utilizadas para a análise de imunoeletroforese de proteínas envolvidas no dano ao DNA e proliferação celular (?H2AX e pAkt, respectivamente), na progressão do ciclo celular (p53, p16 e ciclina A), na captação de glicose (HIF-1a e GLUT1) e proteínas envolvidas na sinalização da morte celular (PARP, Bax, Bcl-xL). Resultados. As quinonas, quando associadas ao ascorbato, causaram um aumento na intercalação e clivagem oxidativa do CT-DNA. Em células MCF-7, a associação com ascorbato diminuiu significativamente a viabilidade celular. As associações juglona/ascorbato e, principalmente Q7/ascorbato, aumentaram os níveis de EROs intracelulares e os danos ao DNA. As três associações testadas causaram diminuição no número de colônias, porém, juglona/ascorbato e Q7/ascorbato foram capazes de inibir a proteína Akt fosforilada (pAkt). Os resultados in vitro foram reproduzidos in vivo. Juglona/ascorbato e Q7/ascorbato inibiram significativamente o desenvolvimento do tumor e aumentaram o tempo de sobrevida dos animais. Os marcadores de estresse oxidativo foram alterados, indicando uma indução na geração de EROs pelos tratamentos, aumentando assim a peroxidação lipídica e a carbonilação de proteínas, danificando o DNA das células do tumor ascítico de Ehrlich e promovendo também, a parada do ciclo celular em G1/S, redução da captação de glicose e morte celular por apoptose. Conclusão. Os resultados apresentados mostraram que o ascorbato de sódio potencializou o efeito antitumoral das quinonas juglona e, principalmente da Q7, também in vivo, evidenciando que estas associações são promissores agentes para o tratamento do câncer.

Abstract : Introduction. Evidences point to the promising antitumor effect of quinones juglone, phenylaminonaphthoquinone-7 (Q7) and phenylaminonaphthoquinone-9 (Q9), especially when associated to ascorbate. Objective. To continue the studies on the antitumor effect of naphthoquinones juglone, Q7 and Q9 associated with sodium ascorbate, as well as to evaluate the best association for cancer treatment from the effects obtained in vivo and in vitro. Methodology. The effects of quinones juglone, Q7 and Q9 associated or not to sodium ascorbate on Calf Thymus-DNA were analyzed. Cell viability, levels of intracellular ROS, damages on DNA and cell proliferation were evaluated in MCF-7 cells (breast carcinoma). Proteins from MCF-7 cell lysate were used in immunoblotting assays to check the integrity of poly (ADP-ribose) polymerase (PARP), gamma-H2AX (?H2AX) and pAkt. The in vivo antitumor activity of quinones associated or not with sodium ascorbate was firstly analyzed by means of survival parameters and tumor growth of mice carrying Ehrlich ascitic tumor. After that, samples of the tumor were collected and analyzed for oxidative stress markers such as: thiobarbituric acid reactive substances (TBARS), carbonylation of protein, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST). Also, of Ehrlich ascitic tumor, DNA damages, cellular cycle arrest, influence in glucose capture and type of cell death induced by the treatments were evaluated. The same samples were used in immunoblotting assays of proteins involved in damage to DNA and cell proliferation (?H2AX and pAkt, respectively), cell cycle progression (p53, p16 and cyclin A) and glucose capture (HIF-1a and GLUT1) and proteins involved in cell death signaling (PARP, Bax, Bcl-xL). Results. When associated to ascorbate, quinones promoted an increase in interleaving and oxidative cleavage of CT-DNA. In MCF-7 cells, the association with ascorbate declined significantly cell viability. The associations juglone/ascorbate and, mainly Q7/ascorbate increased levels of intracellular EROs and damage to DNA. The three assessed associations promoted the decrease in the number of colonies, however, julone/ascorbate and Q7/ascorbate were able to inhibit the protein pAkt. The in vitro results were in vivo reproduced. Juglone/ascorbate and Q7/ascorbate significantly inhibited the growth of the tumor and increased survival time of the animals. The oxidative stress markers were altered pointing to an induction in the generation of ROS by these treatments, thereby increasing lipid peroxidation and protein carbonylation, damaging DNA of cells of Ehrlich ascitic tumor also promoting, cellular cycle arrest in G1/S, reduction of glucose capture and cellular death by apoptosis. Conclusion. The results presented here showed that the sodium ascorbate potentialized the antitumor effect of juglone, especially Q7, also in vivo, evidencing that those associations are promising agents for cancer treatment.

Poly-aryl, also known as polyphenylene compounds are a class of dendrimer, which contain a large number of aromatic rings. They are of interest because they display restricted rotation of their stearically congested aromatic rings. These extended structures have the potential to act as precursors for even larger aromatic systems and have many applications including electronic devices, drug delivery and catalysis. A total of 23 novel poly-aryl compounds have been examined using single crystal X-ray diffraction and a number of structural patterns have emerged. Six of the compounds contain alkynes and it has been observed that their conformation is governed by a combination of conjugation between the alkyne and aryl groups and inter-molecular interactions. In the more extended poly-aryl compounds steric congestion rules out any possibility of conjugation between the rings and their conformation is governed by intra-molecular non-bonded interactions in the core of the molecules and by inter-molecular interactions in their periphery. Where possible, solution NMR measurements were carried out on the poly-aryl compounds and confirmed that the solution structures are in agreement with those obtained from individual crystal.

Solubility prediction usually refers to prediction of the intrinsic aqueous solubility, which is the concentration of an unionised molecule in a saturated aqueous solution at thermodynamic equilibrium at a given temperature. Solubility is determined by structural and energetic components emanating from solid-phase structure and packing interactions, solute–solvent interactions, and structural reorganisation in solution. An overview of the most commonly used methods for solubility prediction is given in Chapter 1. In this thesis, we investigate various approaches to solubility prediction and solvation model development, based on informatics and incorporation of empirical and experimental data. These are of a knowledge-based nature, and specifically incorporate information from the Cambridge Structural Database (CSD). A common problem for solubility prediction is the computational cost associated with accurate models. This issue is usually addressed by use of machine learning and regression models, such as the General Solubility Equation (GSE). These types of models are investigated and discussed in Chapter 3, where we evaluate the reliability of the GSE for a set of structures covering a large area of chemical space. We find that molecular descriptors relating to specific atom or functional group counts in the solute molecule almost always appear in improved regression models. In accordance with the findings of Chapter 3, in Chapter 4 we investigate whether radial distribution functions (RDFs) calculated for atoms (defined according to their immediate chemical environment) with water from organic hydrate crystal structures may give a good indication of interactions applicable to the solution phase, and justify this by comparison of our own RDFs to neutron diffraction data for water and ice. We then apply our RDFs to the theory of the Reference Interaction Site Model (RISM) in Chapter 5, and produce novel models for the calculation of Hydration Free Energies (HFEs).

碩士
長庚大學
生物醫學研究所
103
The spindle-assembly checkpoint (SAC) monitors if chromosomes are attached properly to spindle microtubules, and helps to prevent chromosome mis-segregation. Damage to the checkpoint can lead to aneuploidy, potentially contributing to tumorigenesis. When all chromosomes are attached to the spindle, cyclin B and securing, the two core mitotic regulators at entry into anaphase, are degraded by APC/Ccdc20, an E3 ubiquitin ligase, and the cell cycle proceeds. Besides these two core proteins, I want to find out other proteins that are degraded when the checkpoint is unsatisfied. In the future, these proteins may be candidates as targets for anti-cancer drugs. To identify which proteins are degraded, I will use stable isotope labeling by amino acids in cell culture (SILAC), which is a technique based on mass spectrometry that detects differences in protein abundance using non-radioactive isotopic labeling. I employ a yeast strain where I can control Cdc20 protein expression by methionine. In this way, I plan to grow the strain in two independent cultures with and without APC/Ccdc20 activity, and in heavy or light amino acids, and use SILAC to identify proteins regulated APC/Ccdc20 activity. I have measured 673 proteins which were up-regulated in cdc20-depleted cells. 19 of the 673 proteins were related to mitosis and be potential candidates as substrates regulated by the APC/Ccdc20 at prometaphase. Four of 19 proteins (Okp1, Ame1, Dam1, Spc25) were related with kinetochore. Okp1 and Ame1 were known that they regulated SAC. In the future, I will test the proteins directly as substrates in vitro in an APCCdc20 enzyme assay.

碩士
國立臺灣大學
微生物學研究所
95
Epstein-Barr virus (EBV) is a gamma-herpesvirus closely associated with human malignant diseases. Our previous study has demonstrated that expression of BGLF4 kinase induces multiple premature mitotic events such as chromosome condensation, nuclear lamina disassembly, and cytoskeleton rearrangement through Cdk1 mimicry. One of the major functions of Cdk1 is to regulate anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase which promotes metaphase to anaphase transition by targeting substrates such as cyclin A, cyclin B, and securin for proteasomal degradation. Previously, co-expression of BGLF4 kinase induced the degradation of a centrosomal protein ART-27 through APC/CCdc20 ubiquitination pathway, suggesting that BGLF4 may regulate the E3 ubiquitin ligase activity of APC/CCdc20. To search for possible mechanisms involved, the physical interaction of Cdc20 and BGLF4 in vivo was demonstrated by co-immunoprecipitation in this study. Furthermore, Cdc20 was phosphorylated by BGLF4 in in vitro IP-kinase assay. In co-transfection assays, Cdc20 protein was stabilized by BGLF4 in a dose dependent manner. Because Cdk1 is known to activate APC/C activity through phosphorylating APC/C subunits, Cdc27 was examined for possible phosphorylation induced by BGLF4, and no obvious molecular weight shift was observed. Additionally, we found that the localization of ART-27 was changed upon BGLF4 coexpression and ART-27 was phosphorylated by BGLF4 in vitro. Taken together, we postulate that BGLF4 might stabilize Cdc20 protein through phosphorylation, which may then result in the activation of APC/CCdc20 activity. However, cyclin B1 degradation was not obvious in the presence of BGLF4, suggesting not all APC/CCdc20 substrates are degraded. Thus BGLF4 may also play a role in recruiting ART-27 to APC/CCdc20 for promoting its degradation. Further investigation will be needed to reveal how ART-27 protein stability contributes to BGLF4 induced premature chromosome condensation.

Human genes OPTN and CCDC3, encoding respectively for optineurin and coiled-coil domain-containing protein 3, are part of the PDB6 locus, a genetic hotspot strongly associated with Paget's disease of bone (PDB), the second most prevalent metabolic bone disease after osteoporosis. OPTN and CCDC3 genes share a head-to-head configuration and partially overlapping sequences, located on opposite strands of this locus. We first defined the molecular structure of the two genes based on the in silico identified mRNAs, which included several alternatively spliced transcripts. The task was performed with the aid of bioinformatics tools and online databases, such as expressed sequence tags (ESTs), AceView gene browser and Splign software; as a result, we obtained a comprehensive map of OPTN and CCDC3, emphasizing the size and position of introns and exons of each transcript. Next, we assessed the activity of CCDC3 and OPTN promoter regions; due to their head-to-head disposition, the two genes share a common regulatory sequence. A putative CCDC3 alternative promoter, located downstream and exclusive for certain CCDC3 transcripts, was identified by analysing the gene structure obtained in silico. The activity of the promoter regions was validated by transient transfecting pGL3 reporter constructs, containing the promoter sequences under analysis, into HEK 293 cells, followed by luciferase assays. Trans-acting regulatory proteins, e.g. transcription factors (TFs), putatively involved in the regulation of the two genes, were identified in silico by analyzing the promoter sequences through bioinformatics software. The analysis revealed several putative TF binding sites, including for NF-κB, a TF known to play a role in the pathogenesis of PDB. Transient co-transfection of HEK 293 cells with pGL3 reporter constructs and transcription factor NF-κB expression vectors, followed by luciferase assays, have been performed in order to confirm their role as trans-regulators of the target promoters, and to unveil the presence of a possible co-regulation.
Os genes humanos OPTN e CCDC3, que codificam respectivamente para optineurina e coiled-coil domain-containing protein 3, fazem parte do locus PDB6, um hotspot genético fortemente associado à doença óssea de Paget (PDB), a segunda doença óssea metabólica mais prevalente após a osteoporose. Os genes OPTN e CCDC3 compartilham uma configuração frente a frente e sequências parcialmente sobrepostas, localizadas em cadeias opostas desse locus. Primeiro, definimos a estrutura molecular dos dois genes com os mRNAs identificados in silico, que incluíam vários transcritos alternadamente unidos. A tarefa foi realizada com o auxílio de ferramentas de bioinformática e bancos de dados on-line, como tags de sequência expressa (ESTs), navegador de genes AceView e software Splign; como resultado, obtivemos um mapa abrangente de OPTN e CCDC3, enfatizando o tamanho e a posição dos íntrons e exons de cada transcrição. Em seguida, avaliamos a atividade das regiões promotoras de CCDC3 e OPTN; devido à sua disposição frente a frente, os dois genes compartilham uma sequência reguladora comum. Um promotor alternativo de CCDC3, localizado a jusante e exclusivo para certos transcritos de CCDC3, foi identificado através da análise da estrutura genética obtida em silico. A atividade das regiões promotoras foi validada por construções repórteres de transfecção pGL3 transitórias, contendo as seqüências promotoras em análise, em células HEK 293, seguidas por ensaios de luciferase. Proteínas reguladoras de ação trans, p. fatores de transcrição (TFs), potencialmente envolvidos na regulação dos dois genes, foram identificados in silico através da análise das seqüências promotoras através do software de bioinformática. A análise revelou vários locais de ligação a TF, incluindo NF-κB, um TF conhecido por desempenhar um papel na patogênese do PDB. A co-transfecção transitória de células HEK 293 com construções repórter pGL3 e vetores de expressão contendo o TF NF-κB, seguidos de ensaios de luciferase, foram realizados para confirmar seu papel como reguladores trans dos promotores alvo e para revelar a presença de um possível co-regulação.

碩士
國立成功大學
生理學研究所
101
Axonal and dendritic growth is important for neuronal differentiation and nerve regeneration. We have identified that SMAP1 and CCDC75 are two genes downstream of nuclear respiratory factor 1 and differentially regulate neurtie outgrowth in human neuroblastoma cells. SMAP1 encodes the GTPase activating protein of Arf6 GTPase and CCDC75 is a novel gene predicted to encode a nuclear protein with the potential of DNA binding. It remains unknown whether these two genes play different roles in axonal and dendritic growth in neurons. Here, we used the development of cultured rat hippocampal neurons as a model to investigate whether these two genes have differential function in axonal and dendritic growth. Rat hippocampal neurons were cultured from embryonic day 18. Semi-quantitative RT-PCR analysis suggested that the mRNA level of Smap1 increased but Ccdc75 decreased from early to later stages. Overexpression of Smap1 decreased the length of axons and dendrites and delayed the initiation of axonal collaterals and dendritic branches. In contrast, overexpression of Ccdc75 increased the number of axonal collaterals and dendritic branches, but has no effects on the length of axons or dendrites. On the other hand, knockdown of Smap1 increased the length of axons and dendrites, but has no effects on the number of axonal collaterals and dendritic branches. In contrast, knockdown of Ccdc75 decreased the number of axonal collaterals and dendritic branches, but has no effects on axonal and dendritic length. Smap1-EGFP fusion protein localized in the cytosol and Ccdc75-EGFP fusion protein localized in the nucleus. Smap1 co-localized with clathrin in cultured rat hippocampal neurons. Overexpression of Smap1 blocked clathrin-dependent endocytosis which was demonstrated by the inhibition of transferrin receptor internalization. These results suggest that Smap1 may inhibit axonal and dendritic elongation through clathin-dependent endocytosis and Ccdc75 may enhance axonal and dendritic branching through an unknown mechanism. These results not only increase our knowledge on the molecular network underlying axonal and dendritic growth, but also provide potential therapeutic targets for neuronal regeneration after injury.

acase@tulane.edu
TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPARγ expression and consequently leading to a drastic decrease of the PPARγ target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.
1
Wenjuan Liao

Zusammenfassung Da die meisten Fälle männlicher Unfruchtbarkeit immer noch idiopatisch bleiben, könnte eine Untersuchung der Mechanismen, in denen Gene der Spermatogenese-Kontrolle involviert sind, helfen, die Gründe besser zu verstehen. Diese Arbeit besteht aus zwei Teilen die zwei verschiedene Aspekte humaner Fertilitätsforschung betreffen. Das erste Projekt betrifft die Analyse des coiled coil domain containing 33 (CCDC33) Proteins und seiner Rolle als peroxisomal testis specific 1 (PXT1) Interaktionspartner. Im zweite Projekt haben wir die Ursache männlicher Unfruchtbarkeit in Mäusen mit multiplen knockouts bestimmt und den Phänotyp anlaysiert. Folgende Gene wurden ausgeknocked: Tnp2, Hist1h1t, Theg, Acr, Creb3l4 and Tex22. Im ersten Teil dieser Arbeit wurden Domänen, die bei der CCDC33 und PXT1-Interaktion beteilgt sind, identifiziert. Die Interaktion dieser beiden Proteine wurde duch Kaczmarek identifiziert (Kaczmarek, 2009). Wir zeigten, dass nicht die coiled coil Domänen für die Interaktion mit PXT1 verantwortlich sind, sondern eine Aminosäuresequenz welche der Sequenz in Leucin-reichen Domänen ähnlich ist. Durch induzierte Mutagenese in der PXT1-Sequenz haben wir Aminosäuresequenzen identifiziert, die für die Interaktion von PXT1 und CCDC33 bedeutend sind. PXT1 ist ein erstes peroxisomales Protein das eine funktionale BH3-ähnliche Domäne enthält, die dafür bekannt ist Apoptose zu induzieren. Aufgrund von Experimenten mit Co-transfizieretn HeLa Zellen konnten wir eine Co-Lokalisation von CCDC33 und PXT1 im Zytoplasma nachweisen. Zudem zeigten Zellen mit Co-Lokalisation beider Proteine eine Reduktion der Apoptoserate im Vergleich zu Zellen die nur mit dem PXT1-Konstrukt transfiziert waren. Dieser Befund könnte suggerieren, dass die physikalische Bindung beider Proteine die PXT1-induzierte Apoptose verhindert. Durch Western Blot und immunhistochemische Experimente konnten wir zeigen, dass das CCDC33 Protein in den Hoden während der Spermatogenese ab dem Spermatocyten- Stadium lokalisiert ist. Sowohl die Interaktion von CCDC33 und PXT1 als auch das Expressionsmuster beider Gene implizieren, dass Ccdc33 in der Kontrolle der Spermatogenese involiert sein könnte. Wir postulieren, dass CCDC33 ein neues peroxisomales Protein sein könnte, welches Apoptose während der Spermatogenes reguliert. Jedoch sind weitere Untersuchungen, u.a. die Analyse von CCDC33 knockout Maus-Modellen, nötig, um feststellen zu können ob Ccdc33 für die männliche Fertilität essentiell ist. Im zweiten Teil dieser Arbeit wird die Analyse von knockout Mäuselinien, in denen 6 keimzellspezifische Gene funktionsunfähig gemacht wurden, beschrieben. In ca. 20% der Mäuse wurde Unfruchtbarkeit festgestellt. In beiden, männlichen fruchtbaren und unfruchtbaren 6xKO Mäusen, ist die Zahl an Spermatozoa mit abnormalen Kopf erhöht und Sperma-Motilität reduziert. Nach Kreuzung von weiblichen wildtyp Mäusen mit männlichen 6xKO Tieren, benötigte das Sperma bemerkenswert längere Zeit um die Oocyte zu befreuchten as im Vergleich zu WT Sperma. Die Unterschiede im Phänotyp der 6xKO Mäuse sind sowohl dem genetischen Hintergrund als auch der Inaktivierung der sechs keimzellspezifischen Gene zuzuschreiben. Um genauer zu untersuchen welche Gene bei der Unfruchhtbarkeit der 6xKO Mäuse involviert sein könnten haben wir ein Transkriptom Assay durchgeführt und die Genexpression in den Hoden von 5xKO Tieren, die fertil waren, mit der in 6xKO Mäusen, die infertil waren, verglichen. Wir haben ein neues Gen, 4933400A11Rik, identifiziert und charakterisiert, welches für die Unfruchtbarkeit männlicher 6xKO Mäuse verantwortlich sein könnte. In WT Mäusen ist 4933400A11Rik in den Keimzellen exprimiert. 4933400A11Rik mRNA wurde ab Tag 16 post partum, wenn primäre Spermatozyten produziert werden, bis zu den ausgewachsenen Spermien detektiert. Es ist jedoch nicht klar, ob die mRNA die in den Spermien entdeckt wurde ein Ergebniss der Expression von 4933400A11Rik sind, oder ob es sich dabei um restliche Moleküle handelt welche während der Spermatogenese nicht abgebaut wurden. 4933400A11RIK weist Sequenzähnlichkeiten zur Aktinfilament capping Proteinfamile auf. Wir zeigten, dass 4933400A11RIK mit F-Aktin capping Protein Untereinheit alpha-1 colokalisiert. Proteine dieser Familie sind bekannt dafür das Wachstum des Zytoskeletts durch Regulation der Aktinfilament-Reorganisation zu kontrollieren. Deregulation der Aktinzytoskelett-Reorganisation in Keimzellen könnte die zugrundeliegende Ursache der männlichen Unfruchtbarkeit in 6x KO Mäusen sein, denn 4933400A11Rik wird in den Hoden fertiler 6xKO Mäuse, aber nicht in den Hoden infertiler 6xKO Mäuse exprimiert. Um die Frage eindeutig zu beantworten, ob das Gen 4933400A11Rik die Fertilität beeinflusst, sollten 4933400A11Rik knockout Mäuse generiert und analysiert werden. Das ´Rescue-Experiment´ könnte ebenso die Involvierung dieses Gens bei männlichen Fertilität bestätigen. Zusammenfassend präsentiert diese Arbeit interessante Erkenntnisse über die potentielle Funktion von CCDC33 bei der Regulation der Spermatogenese. Außerdem zeigen wir kumulierte Effekte von 6 keimzellspezifischen Genen und einem neuen Gen, 4933400A11Rik, welches für die weitere Forschung männlicher Unfruchtbarkeit interessant sein könnte.

博士
國立中興大學
生物醫學研究所
106
Abstract Epithelial-to-mesenchymal transition (EMT) has, in recent years, emerged as an important tumor cell behavior associated with high metastatic potential and drug resistance. Interestingly, protein SUMOylation and hepatocyte growth factor could respectively reduce the effect of small molecule inhibitors on tyrosine kinase activity of mutated epidermal growth factor receptor of lung adenocarcinomas (LADC). The actual mechanism behind this is yet to be resolved. We therefore used immunohistochemistry to stain proteins in LADC specimens. Protein expression was confirmed by Western blotting. In vitro, expression of proteins was determined by Western blotting and immunocytochemistry. Levels of mRNA and circular RNA were determined by reverse transcription-polymerase chain reaction. Our results showed that SUMOylation-activating enzyme subunit 2 (SAE2) and cirRNA CCDC66 (coiled-coil domain containing 66) were highly expressed in LADC. Expression of SAE2 was mainly regulated by epidermal growth factor receptor (EGFR); however, expression of cirRNA CCDC66 was positively regulated by oncogenes FAK and c-Met, but negatively modulated by nicotinic acetylcholine receptor 7-alpha (nAchR-α7). EGFR resistant H1975 also highly expressed cirRNA CCDC66. Immediate response of hypoxia increased phosphorylated c-Met, SAE2 and EMT. Either activation of FAK or silencing of nAchR-α7 increased cirRNA CCDC66. These experimental outcomes suggest that HGF/c-Met regulates expression of SAE2 and cirRNA CCDC66 to increase EMT and drug resistance of LADC cells. Multimodality drugs concurrently aiming at these targets would probably provide more benefits for cancer patients. It is worth noting that nicotine, through nAChR-α7, activates HGF expression in LADC cells, suggesting an important role of nAChR-α7 in affecting cell proliferation, and motility. Overexperssion of nAChR-α7 was also detected in uterine cervical and gastric cancer. Knockdown of nAChR-α7 reduced drug resistance, and EMT confirmed the significance of nAChR-α7, and validated that nAchR-α7 could be a potential target of cancer therapy as well.

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
Os vertebrados apresentam uma organização corporal complexa com três eixos: eixo antero-posterior, o eixo dorso-ventral e o eixo esquerda-direita. As assimetrias nos eixos antero-posterior e dorso-ventral são facilmente identificáveis exteriormente. No entanto, no que diz respeito ao eixo esquerda direita, as diferenças são notadas unicamente na disposição interna dos órgãos. No Homem, o coração está deslocado para o lado esquerdo, assim como o estômago e o baço, enquanto que o fígado e a vesícula biliar ficam do lado direito. Também os pulmões, devido à posição do coração, apresentam assimetria no número de lóbulos. Esta disposição (situs solitus) é determinada cedo durante o desenvolvimento embrionário e mantem-se conservada nos vertebrados. A diferenciação do eixo esquerda-direita é precedida por assimetrias na expressão de genes no organizador esquerda-direita (LRO, na sigla em inglês). O LRO é uma estrutura transiente localizada no final da notocorda, que no peixe-zebra é chamada de vesícula de Kupffer (KV). Esta vesícula é formada por células, cada uma com um cílio diferenciado na sua membrana apical. A maioria destes cílios são móveis e com o seu movimento coordenado produzem um fluxo direcionado para o lado esquerdo do LRO. Esta assimetria no fluxo é depois transferida para uma assimetria na expressão de genes nos tecidos à volta do LRO. Inicialmente há dois genes que são expressos de forma simétrica à volta do LRO: nodal e cerl2. A forma como o fluxo consegue controlar a expressão de nodal e cerl2 ainda não é conhecida, mas foram propostas duas hipóteses que a tentam explicar. A primeira propõe que o fluxo gerado pelos cílios móveis transporta substâncias secretadas pelo LRO e as concentra do lado esquerdo, levando à assimetria na expressão nodal e cerl2 verificada posteriormente. A outra hipótese propõe que os cílios móveis criam o fluxo de fluído que, no lado esquerdo, vai causar a deflação dos cílios imóveis, estimulando os seus mecanorecetores e induzindo uma via de ativação dependente de Ca2+ que culmina na expressão assimétrica de nodal e cerl2. No entanto, apesar dos esforços, ambas as hipóteses têm sido questionadas e o exacto mecanismo de transdução da informação permanece desconhecido. Após a ação do fluxo, que é sentido pelas células que rodeiam o LRO, a expressão de cerl2 é inibida no lado esquerdo. A inibição da expressão de cerl2 (inibidor de nodal) permite uma maior expressão de nodal e consequentemente a ativação da cascata de sinalização Nodal-Lefty-Pitx2. Pitx2 é a proteína efetora da cascata iniciada por Nodal, sendo apontada como a responsável por induzir o fenótipo “esquerdo” nas células do lado esquerdo da mesoderme lateral (L-LPM). Os cílios, que podem ser móveis ou imóveis (primários), estão presentes em quase todos os tipos células nos vertebrados e por isso, são determinantes quer no período de desenvolvimento embrionário quer na idade adulta. Quando o seu funcionamento fica comprometido pode desencadear várias doenças, como por exemplo a doença do rim poliquístico (PKD, na sigla em inglês) ou síndromes somáticas associadas a polidactilia, malformações neurológicas ou obesidade, no caso de alterações associadas aos cílios primários. Quando são os cílios móveis afetados, as alterações manifestam-se através de infertilidade, infeções respiratórias recorrentes e situs inversus que caracterizam a uma condição chamada discinesia ciliar primaria (PCD). A PCD afecta 1 em cada 10 000 nascimentos e é uma das causas de defeitos cardíacos congénitos. A presença de heterotaxia, condição na qual, pelo menos um órgão está no lado oposto ao que seria de esperar, aumenta 200 vezes a prevalência destes defeitos congénitos. A PC pode ser causada por mutações que alterem ou inibam a produção de qualquer um dos componentes necessários à montagem das estruturas essenciais para o movimento dos cílios, como o par central, os raios, o complexo regulador da nexina, os braços interno e externo de dineínas ou outras proteínas citoplasmáticas que participam na montagem de componentes ciliares. Este trabalho foca-se na proteína ccdc40, que é um dos componentes do braço interno de dineínas. Esta proteína é também responsável pela poliglutamilação dos microtúbulos contribuindo para a sua estabilização. Além disso, a proteína Ccdc40 forma um complexo com outra, a Ccdc39, e atua como uma régua molecular. Este complexo permite que os raios dos cílios apenas se liguem no local devido ao longo dos microtúbulos, isto é, a cada 96 nm. Na sua ausência, estes raios continuam a ligar-se aos microtúbulos, mas em locais onde isso não era suposto acontecer. Em pacientes com PCD portadores de alterações neste gene, foram identificados locais da proteína onde as mutações são mais frequentemente encontradas: as mutações c.248delC e c.3129delC alteram a grelha de leitura enquanto que as outras (c.2440 C>T, c.961 C>T e c.1345C>T) produzem proteínas truncadas. Neste trabalho, pretendeu-se criar um peixe-zebra mutante que produza a proteína Ccdc40 truncada e que possa servir como modelo de doença para estudar o impacto das mutações mais perto do terminal N da proteína (c.248delC e c.961 C>T). Estas mutações próximas da extremidade N representam cerca de 50% dos casos de mutações identificadas neste gene e produzem proteínas sem o domínio helicoidal (coiled-coil). Para produzir este mutante usou-se o sistema CRISPR-Cas9. Este sistema foi adaptado a partir de um mecanismo de defesa descoberto em bactérias e permite direcionar a Cas9 (DNase) para um local específico do genoma, usando uma sequência guia de RNA complementar ao local escolhido. Uma vez clivado o DNA, a célula inicia o processo de reparação através da junção das extremidades não homólogas (NHEJ), processo este que é propenso à inserção ou remoção de bases azotadas, levando à ocorrência de mutações que podem inativar a proteína. Neste caso, o local escolhido como alvo da Cas9 foi a zona do genoma que codifica para o aminoácido 116 da proteína ccdc40 no peixe zebra. Esta região é homóloga da região do genoma humano onde ocorre a mutação c.961 C>T. Uma mutação nonesense ou uma alteração da grelha na leitura neste local impede totalmente a tradução do domínio em hélice, o que causa alterações na montagem da estrutura interna dos cílios e consequentemente na sua mobilidade. De forma a poder estudar e futuramente validar o fenótipo resultante da injeção do morfolino bloqueador da tradução, foram analisados a posição do coração, fígado e pâncreas assim como defeitos na cauda e aparecimento de edema cardíaco, em embriões injetados com o morfolino. Os resultados mostraram que, independentemente da concentração injetada, os embriões apresentam defeitos relativos à posição do coração (coração à direita ou ao centro), fígado e pâncreas, assim como curvaturas na cauda e aparecimento de edema por volta dos 3dpf. Estes resultados, irão futuramente ser comparados com o fenótipo observado no mutante produzido, validando ou não este morfolino. Além disso, este mutante permitirá estudar as alterações estruturais e funcionais dos cílios em que a proteína Ccdc40 apresenta uma mutação próxima da extremidade N e compará-las com a estrutura dos cílios de outa linha mutante já existente, lok, em que a proteína é mutada próximo da extremidade C e ainda possui parte do domínio helicoidal. Futuramente, este mutante poderá ser útil para testar terapia génica direcionada à correção desta mutação que poderá mais tarde ser reproduzida em células humanas recolhidas de pacientes com PCD com o intuito de serem reimplantadas, possibilitando uma forma de tratamento que possa minimizar os efeitos da PCD no aparelho respiratório.
On the surface, vertebrates seem to have a bilateral symmetry. However, the disposition of the internal organs, such as the heart and liver says otherwise. The left-right axis differentiation is preceded by asymmetries in the gene expression pattern in the tissues around the left-right organiser (LRO). The LRO (called Kupffer’s vesicle in zebrafish) is a transient structure localized at the end of the notochord that is formed by cells contain a cilium protruding from its apical membrane. Most of these cilia are motile and their beating movement creates a fluid flow towards the left side of the LRO. In a way not yet fully understood, the cells surrounding the LRO sense the fluid directionality and trigger asymmetric expression of nodal and cerl2. The expression of nodal on the left side of the LRO triggers the Nodal-Lefty-Pitx2 pathway that leads to the typical positioning of the internal organs recognised as situs solitus. Besides the laterality establishment, the respiratory epithelium, the ependymal cells lining the brain ventricles, the oviducts epithelium and the sperm cells also relay on motile cilia to function properly. When motile cilia function is impaired, this sequence of events is not guaranteed and patients may suffer from heart congenital malformations bronchiectasis and infertility that characterize primary ciliary dyskinesia (PCD). PCD is caused by mutations in a vast number of genes that codify for cilia components. One of those genes is called CCDC40 and codifies for a protein necessary for the assembly of the inner dynein arms (IDA) and the nexin-dynein regulatory complexes (N-DRC). In this work, we generated a ccdc40-/- zebrafish mutant (116 aa) using a CRISPR-Cas9 approach. Due to the long zebrafish maturation period, it was not possible to study the mutant phenotype. Instead, we studied the phenotype of embryos injected with a translation-blocking morpholino (MO). We noted that the organ situs was altered in 40 to 70% of the cases, (depending on the amount of MO-injected) and the developing of tail malformations (not related to the injected MO amount). The ccdc40-/- zebrafish mutant was designed to replicate a human mutation and can be used to test gene editing approaches in an attempt to develop a PCD treatment.