Dissertationen zum Thema „Tbc1D3“

Les malformations du développement du cortex sont associées à des troubles de la prolifération des progéniteurs et de la migration neuronale. Les glies radiaires basales (bRGs), un type de progéniteur, sont limités dans les espèces lissencéphaliques mais abondants dans les cerveaux gyrencéphaliques. Le gène LIS1, codant pour un régulateur de la dynéine, est muté dans la lissencéphalie humaine. LIS1 a un rôle dans la division cellulaire et la migration neuronale. Dans cette étude, nous avons généré des cellules bRG-like dans le cerveau embryonnaire murin, pour étudier le rôle de Lis1 dans leur production. Ceci fut réalisé par électroporation in utero du gène hominoïde-spécifique TBC1D3 au jour embryonnaire (E) 14.5. Nous avons confirmé que l’expression de TBC1D3 dans des cerveaux WT induit un grand nombre de cellules bRG-like basales. Puis, nous avons étudié la production des bRGs-like dans des cerveaux murins hétérozygotes pour Lis1. Nos résultats novateurs montrent que la déplétion de Lis1 à partir de E9.5 empêche la production de cellules bRG-like induites par TBC1D3. La déplétion de Lis1 change l’orientation du fuseau mitotique, accroit le nombre de mitoses abventriculaires et altère l’expression de N-Cadhérine. Nous concluons que la perturbation du dosage de Lis1 pourrait perturber le nombre et la position corrects des progéniteurs, contribuant à la pathogenèse de Lis1
Human cortical malformations are associated with progenitor proliferation and neuronal migration abnormalities. Basal radial glia (bRGs), a type of progenitor cells, are limited in lissencephalic species (e.g. the mouse) but abundant in gyrencephalic brains. The LIS1 gene coding for a dynein regulator, is mutated in human lissencephaly, associated also in some cases with microcephaly. LIS1 was shown to be important during cell division and neuronal migration. Here, we generated bRG-like cells in the mouse embryonic brain, investigating the role of Lis1 in their formation. This was achieved by in utero electroporation of a hominoid-specific gene TBC1D3 at mouse embryonic day (E) 14.5. We first confirmed that TBC1D3 overexpression in WT brain generates numerous Pax6+ bRG-like cells that are basally localized. Second, we assessed the formation of these cells in heterozygote Lis1 mutant brains. Our novel results show that Lis1 depletion in the forebrain from E9.5 prevented subsequent TBC1D3-induced bRG-like cell amplification. Lis1 depletion changed mitotic spindle orientations at the ventricular surface, increased the proportion of abventricular mitoses, and altered N-Cadherin expression, altering TBC1D3 function. We conclude that perturbation of Lis1/LIS1 dosage is likely to be detrimental for appropriate progenitor number and position, contributing to lissencephaly pathogenesis

Nob1 (New Zealand obese 1) bezeichnet einen Adipositas-QTL auf Chr. 5 der Maus (LODBMI >3,3), der in einem Rückkreuzungsexperiment der Mausstämme NZO (adipös) und SJL (schlank) identifiziert wurde. Um Kandidatengene für Adipositas zu finden, wurden mehr als 300 Nob1-Transkripte mit Hilfe von Genexpressionsanalysen auf Unterschiede in stoffwechselrelevanten Geweben zwischen beiden Mausstämmen untersucht. Sieben Gene zeigten eine differentielle Expression: 2310045A20Rik, Tbc1d1, Ppp1cb, Mll5, Insig1, Abhd1 und Alox5ap. Die codierenden Bereiche dieser Gene wurden anschließend auf Sequenzunterschiede zwischen NZO und SJL untersucht. Nur im Gen Tbc1d1, das im Peak-Bereich des Nob1 lokalisiert ist, wurde eine SJL-spezifische Deletion von sieben Basen detektiert, die zu einer Leserasterverschiebung und einem vorzeitigen Abbruch des Proteins in der funktionellen Rab-GAP-Domäne führt (Loss-of-Function-Mutation). Interessanterweise wurde eine Variante von TBC1D1 (R125W) in Kopplungsanalysen mit Adipositas beim Menschen assoziiert (Stone et al., 2006). TBC1D1 zeigt eine hohe Homologie zu TBC1D4 (AS160), das im Insulinsignalweg eine wichtige Rolle spielt. In 17 weiteren Genen im Peak-Bereich des Nob1 wurde keine weitere SJL-spezifischen Mutation detektiert. Bei NZO-Tieren erfolgte die Tbc1d1-mRNA-Expression vorwiegend in glycolytischen Fasern des Skelettmuskels. Zudem wurden zwei gewebsspezifisch exprimierte Tbc1d1-Isoformen identifiziert, die sich durch alternatives Splicen der Exone 12 und 13 unterscheiden. Die im Rahmen dieser Arbeit gefundenen Ergebnisse machen Tbc1d1 zu einem plausiblen Kandidatengen für den Nob1-QTL. Welche Funktion Tbc1d1 im Glucose- und Fettstoffwechsel des Skelettmuskels hat, muss in weiteren Analysen untersucht werden.
Nob1 (New Zealand obese 1) has been identified as an obesity QTL on chromosome 5 (LODBMI >3,3) in a backcross experiment of obese NZO and lean SJL mice. To identify candidate genes for obesity expression profiling experiments with RNA from metabolic tissues were performed with more than 300 Nob1-genes. Seven genes showed differences in mRNA expression levels between both strains: 2310045A20Rik, Tbc1d1, Ppp1cb, Mll5, Insig1, Abhd1, and Alox5ap. Sequencing of the coding regions of these genes revealed a SJL-specific deletion of seven basepairs in the Tbc1d1 gene that is located in the peak region of Nob1. This mutation leads to a frameshift resulting in a truncated protein that lacks the important Rab-GAP-domain (Loss-of-Function-mutation). Interestingly, linkage analysis of the R125W-variant of TBC1D1 has been recently associated with human obesity. TBC1D1 shows high homology to TBC1D4 (AS160) that plays an important role in the insulin signaling pathway. No other SJL-specific mutations were detected in 17 further genes in the Nob1 peak region. In NZO mice Tbc1d1 mRNA is predominantly expressed in glycolytic fibres of skeletal muscle. Two isoformes were identified differing in alternative spliced exons 12 and 13 and showing a tissue specific mRNA expression. The results presented in this work make Tbc1d1 a very feasible candidate gene to be causal for Nob1. The function of Tbc1d1 in the metabolism of carbohydrates and fat has yet to be analyzed.

Dissertação para obtenção do grau de Mestre em Biologia Molecular em Saúde
O centrossoma é um organito essencial nos eucariotas sendo o principal centro organizador de microtúbulos nas células animais. É composto por um par de centríolos e rodeado por uma matriz pericentriolar. Em células em interfase, os centrossomas estão envolvidos na nucleação/organização dos microtúbulos, no posicionamento dos organitos, e.g. o complexo de Golgi, no estabelecimento da polaridade e ainda na migração e adesão, por sua vez em mitose facilitam a formação dos fusos mitóticos.
Estudos realizados pelo nosso grupo, identificaram uma nova proteína humana, que contem o domínio TBCC (TBCCD1), a qual está relacionada com o cofator C da tubulina, o qual participa na via de folding da tubulina apresentando uma atividade GAP (GTPase activating protein) para a β-tubulina. O TBCCD1 é um componente centrossomal, localizando-se também na zona mediana do fuso, corpo médio e corpos basais/zona de transição de cílios primários e móveis. O silenciamento do TBCCD1 em células RPE-1 provocou um aumento acentuado da distância núcleo-centrossoma, um atraso no ciclo celular, desorganização do complexo de Golgi e baixa eficiência para formar cílios primários. Através de técnicas de análise mutacional identificou-se o domínio mínimo necessário à localização do TBCCD1 no centrossoma, o qual corresponde aos 20 primeiros resíduos de aminoácidos da sua região N-terminal.
O splicing alternativo do pré-mRNA é um passo crítico para a expressão de genes sendo a principal fonte para a diversidade de proteínas nos eucariotas superiores. Atualmente pensa-se que ocorre em mais de 90% dos genes humanos. A proteína TBCCD1 humana é codificada por um gene localizado no cromossoma 3 (3q27.3) e apresenta a sua região codificante interrompida por 7 intrões. O presente estudo permitiu verificar que este gene origina três transcritos diferentes pelo processo de splicing alternativo. Um destes transcritos resulta do facto que existem dois primeiros exões alternativos, que originam duas proteínas putativas diferindo nos primeiros resíduos de aminoácidos da sua N-terminal. Esta sequência de aminoácidos alternativos corresponde no TBCCD1 ao domínio envolvido na sua localização centrossomal. De facto, as duas novas variantes apresentam uma localização citoplasmática não se localizando no centrossoma.

Autophagy is a bulk degradation process characterised by the formation of double membrane vesicles called autophagosomes. Autophagosomes derive from a small precursor structure called phagophore, which is expanded to enclose a portion of cytoplasm or organelle, and finally fuses with the endo-lysosomal system to acquire degradative capacity. Autophagy is often studied as a response to starvation, since the degraded components can be re-used for biosynthetic pathways. However, in multicellular organisms, it has many additional functions in tissue homeostasis, during development, in infection and immunity, and in programmed cell death. Regarding autophagy many questions remain such as the origin of the autophagosomal membrane, the mechanism of phagophore extension and many of the signalling pathways that lead to autophagosome assembly. To better understand the membrane trafficking events involved in autophagosome formation I did an over-expression screen for Rab GTPase activating proteins (RabGAPs) inhibiting this process. RabGAPs act inhibitory on Rab GTPases, which are major regulators of intracellular membrane traffic. I have identified 11 RabGAPs that inhibit autophagosome formation, one of which is TBC1D14, which binds to ULK1, an important kinase for initiation of autophagy. TBC1D14 also binds to Rab11 and tubulates recycling endosomes when over-expressed. I found that both ULK1 and another essential autophagy protein mAtg9 localise to recycling endosomes under normal conditions. I propose that recycling endosomes can signal through both ULK1 and mAtg9 to initiate autophagosome formation upon starvation and may be important in monitory cellular nutrient status, and/or provide membrane to autophagosomes. Investigating the role of ULK1 in vivo, I found that ULK1 knock-out mice are able to undergo basal autophagy as assessed by LC3 lipidation in various tissues. However, ULK1 knock-out mice have less CD4 and CD8-positive mature T cells and their response to TCR stimulation is impaired compared to T cells from wild type animals.

La maladie de Paget est caractérisée par un remodelage osseux anarchique débutant par une phase de résorption excessive suivie d’une phase de formation désordonnée. Comme les ostéoclastes sont recrutés en plus grand nombre et sont hyperactifs aux sites affectés par la maladie, cette cellule a été incriminée dans ce désordre osseux. Le phénotype de l’ostéoclaste pagétique comporte de plus un défaut du processus autophagique, de même qu'une résistance à l'apoptose, dont les mécanismes restent mal connus. Certains facteurs génétiques et environnementaux contribuent en partie au phénotype, mais d'autres facteurs pourraient y être associés. Des travaux du laboratoire ont mis en évidence six événements de l’épissage alternatif associés à la maladie de Paget. Parmi les gènes identifiés, le gène TBC1D25 et ses deux isoformes connus n’ont jamais été étudiés dans l’ostéoclaste. Le gène TBC1D25 possède un domaine hautement conservé TBC régulant l’activité des petites GTPases Rabs dans le transport vésiculaire et un domaine LIR liant la protéine LC3 durant le processus de l’autophagie. Ces domaines fonctionnels se retrouvent seulement dans l’isoforme long. L’hypothèse de recherche est que l’altération de l’épissage alternatif du gène TBC1D25 dans les ostéoclastes pagétiques explique en partie le phénotype de la cellule. Le changement dans le ratio de l’expression des isoformes affecterait le processus de l’autophagie, en plus d’affecter la principale fonction de l’ostéoclaste, la résorption osseuse. L’objectif principal de l’étude est de caractériser l’expression et la fonction de TBC1D25 dans les ostéoclastes humains. Des ostéoclastes humains différenciés à partir de monocytes fœtaux ont été utilisés pour l’étude de la fonction de TBC1D25 dans l’autophagie, l’apoptose et la résorption osseuse. Les travaux ont permis de localiser les protéines dans l’ostéoclaste dans des conditions maintenant un niveau basal de l’autophagie et dans des conditions induisant l’autophagie. L’interaction entre TBC1D25 et Rab34 a été confirmée pour la première fois dans les ostéoclastes. De plus, une variation de cette interaction a été observée dans les différentes conditions modulant le niveau d’induction de l'autophagie. Les résultats préliminaires montrent une augmentation du ratio LC3II/LC3I lors de la diminution de l’expression de TBC1D25 dans des conditions augmentant l’induction de l'autophagie. Par contre, aucun effet a été observé sur la résorption osseuse ou sur l'apoptose lors de la diminution de l’expression de TBC1D25. En conclusion, les résultats préliminaires montrent que TBC1D25 préviendrait l’augmentation du ratio LC3II/LC3I dans l’ostéoclaste soit en inhibant l’induction de l’autophagie ou en favorisant la dégradation des autophagosomes par l’entremise de son action sur Rab34.
Abstract : Paget’s disease of bone (PDB) is characterized by increases in bone turnover starting with excessive resorption followed by disorganized bone formation. Because the initial phase of PDB involves excessive bone resorption, osteoclasts have been identified as the cells primarily affected in PDB. Pagetic osteoclasts are overactive, resistant to apoptosis and exhibit defects in autophagy, but the mechanisms involved are still unclear. While genetic and environmental factors associated with PDB may partially account for the osteoclast phenotype, other genetic contributors have been identified. Recent work from our laboratory has identified six alternative splicing events associated with PDB. Among those genes, TBC1D25 and its two known isoforms have never been studied in osteoclasts. The two functional domains of TBC1D25 (TBC and LIR) are only present in the long isoform. The highly conserved TBC domain regulates small Rab GTPases in vesicular transport and the LIR domain interacts with LC3 during autophagy. Our research hypothesis is that altered alternative splicing of TBC1D25 in pagetic osteoclasts could contribute to phenotype. Differential isoform expression could affect osteoclast autophagy and bone resorption. The aim of the study is to characterize the expression and function of TBC1D25 proteins in human osteoclasts. Osteoclasts differentiated from cord blood monocytes were used to investigate the function of TBC1D25 in autophagy, apoptosis and bone resorption. First, the localization of the protein has been characterized in conditions maintaining basal autophagy and in rapamycin-induced autophagy. Interactions between TBC1D25 and Rab34 have been observed for the first time in osteoclasts. Moreover, changes in the interaction were observed with autophagy induction. Preliminary results suggest increases in LC3II/LC3I ratio with decreasing TBC1D25 expression when autophagy induction is stimulated. On the other hand, preliminary results showed that decreased expression of TBC1D25 did not affect bone resorption, nor apoptosis. In conclusion, preliminary results show that in osteoclasts, TBC1D25 could prevent the increase of LC3II/LC3I ratio by inhibiting autophagy induction or by promoting the clearance of autophagosomes through its action on Rab34.

L’autophagie est un processus de dégradation et de recyclage des composés cellulaires. Ce mécanisme est nécessaire que ce soit à l’état basal pour éliminer des agrégats protéiques ou des organites endommagés ou en condition de stress, tels que la carence nutritionnelle, l’hypoxie ou encore des traitements anticancéreux. De ce fait, l’autophagie est un processus essentiel à la survie ainsi qu’au maintien de l’homéostasie cellulaire. Connaître les joueurs et comprendre les mécanismes de régulation de l’autophagie sont donc importants. Les GTPases RABs sont des régulateurs importants de ce processus. Celles-ci agissent comme des interrupteurs moléculaires permettant d’exécuter rapidement des fonctions dans la cellule. Les RABs sont activées par des Guanine Nucleotide Exchange Factors (GEF) alors que les GTPase Activating Proteins (GAP) accélèrent la désactivation de la RAB. RAB21 est essentielle dans les étapes tardives de l’autophagie. En effet, RAB21 est activée par la carence nutritionnelle, via sa GEF MTMTR13, et permet le trafic d’une SNARE requise pour le flux autophagique. Lors d’une carence prolongée, l’activité de RAB21 diminue rapidement, suggérant ainsi le rôle d’une GAP dans cette régulation négative. Toutefois, aucune GAP pour RAB21 n’a été identifiée jusqu’à maintenant. Un criblage génétique chez la drosophile a permis d’identifier quelques candidats. Suite à des essais d’interactions protéiques, il s’est avéré que seule la GAP TBC1D25 interagissait avec RAB21. De plus, cette interaction est augmentée en fonction de la carence nutritionnelle. Des immunofluorescences par microscopie confocale ont révélé que l’interaction RAB21-TBC1D25 était située en partie au niveau des endosomes précoces. Par ailleurs, une activation prolongée de RAB5, située sur les endosomes précoces, inhibe l’interaction RAB21-TBC1D25. De plus amples expériences devront être réalisées afin d’expliquer ces résultats. Dans un autre ordre d’idée, RAB21 est surexprimée dans les cellules ayant un flux autophagique élevé ainsi que dans certaines tumeurs de cancer du côlon (données non publiées du laboratoire). L’expression de Tbc1d25 dans ces mêmes tumeurs ne semble pas augmentée, indiquant que TBC1D25 pourrait être un inhibiteur autophagique spécifique aux cellules ayant un flux autophagique élevé. À la lumière des résultats obtenus, TBC1D25 semble être une GAP pour RAB21 qui permet sa régulation négative suivant l’activation de l’autophagie induite par la carence nutritionnelle.
Abstract : Autophagy is defined as the lysosomal degradation and recycling of cellular constituents. At basal levels, autophagy eliminates protein aggregates or damaged organelles. In condition of stress, such as in condition of nutritional deficiency, hypoxia or cancer treatments, autophagy allow cells to adapt and survive. Therefore, autophagy is an essential system required for survival and maintenance of cellular homeostasis. It is thus essential to identify the cellular entities and mechanisms regulating this process. RAB GTPases were identified as master regulators of autophagy. These particular proteins act as molecular switches for the rapid execution of cellular responses. RABs are activated by Guanine Nucleotide Exchange Factors (GEF) whereas GTPase Activating Proteins (GAP) accelerates RAB deactivation. RAB21 is essential in the late stages of autophagy. Indeed, RAB21 is activated by nutritional deficiency, via its GEF MTMTR13, to allow trafficking of a SNARE required for autophagic flux. During starvation, RAB21 is deactivated which suggest that a GAP could negatively regulate RAB21 activity. However, to date no GAP for RAB21 has been identified. An eye modifier genetic screen in Drosophila was performed to identify potential RAB21 GAPs and some candidates were identified. As a result of this screen, the GAP TBC1D25 was identified as interacting with RAB21. Moreover, this interaction was increased by starvation. Proximity ligation assays revealed that the RAB21-TBC1D25 interaction partially localized at early endosomes. Moreover, prolonged activation of RAB5, located at early endosomes, inhibited RAB21-TBC1D25 interaction. Further experiments will be carried out to explain these results. With respect to the roles of autophagy in cancer, RAB21 was shown to be overexpressed in cells with high autophagic flux as well as in some colon cancer tumors. Importantly, the expression of Tbc1d25 in these same tumors does not appear to be increased, indicating that TBC1D25 could be an autophagic inhibitor specific to cells with a high autophagic flow. My work suggests that TBC1D25 could function as a GAP to negatively regulate RAB21 activity in condition of prolonged starvation.

Cushing's syndrome and glucocorticoid therapy lead to central obesity, insulin resistance, and symptoms of altered energy regulation similar to those observed in the metabolic syndrome. We hypothesized that excess glucocorticoids alter energy sensing/signaling in skeletal muscle through mediation of the LKB1/AMPK signaling pathway. To test this hypothesis, three 100 mg pellets of corticosterone were implanted subcutaneously in each of nine rats for two weeks. Responses were compared with sham operated controls fed ad libitum or food restricted to produce the body weights similar to the treatment group rats. After the treatment period, animals were anesthetized and the right gastrocnemius-plantaris and soleus were removed for analysis. After tibial nerve stimulation for 5 min, the left gastrocnemius-plantaris and soleus were also removed. We assessed AMPK activity and subunit expression, as well as several metabolic indicators including ATP, creatine phosphate, creatine, glycogen, and malonyl-CoA levels in rested and stimulated gastrocnemius-plantaris and soleus muscles. We found that high levels of glucocorticoids decreased AMPKγ3 subunit expression in the gastrocnemius-plantaris. We also observed reduced AMPKα2 activity in the stimulated gastrocnemius-plantaris, but not the soleus; and that this decreased activity corresponded to a significant reduction in phosphorylated TBC1D1, a protein involved in signaling GLUT-4 translocation. Finally, in the gastrocnemius-plantaris, we also noted an increase in glycogen stores in the hypercorticosteronemic rats. Our data suggest that altered energy sensing/signaling associated with high levels of glucocorticoids may be due in part to inhibition of AMPKα2 activity and the high energy state produced by increased glycogen stores. We also conclude that high levels of glucocorticoids decrease the levels of AMPKγ3 and diminish insulin/contraction signaling through phosphorylated TBC1D1.

GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.

Supplying a proper amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably complicated at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. It is known that this sequential step is performed by the tubulin folding cofactor pathway, comprising of a specific set of regulatory proteins - cofactors A to E. Following detailed biochemical studies in vertebrates, studies have been carried out in in vivo models using S. pombe, where the pathway is absolutely essential for viability and each component is evolutionarily conserved. The significance of the data and the possibility of the cofactors playing additional roles led us to study Tbc1, the cofactor C orthologue, which we isolated as a high-copy suppressor of α2-tubulin mutants (atb2-983). Previous work from others and our own lab indicated that Arl2/Alp41 is a GTP binding protein that would be regulated by the GAP, cofactor C/Tbc1. In order to pursue the in vivo roles of Tbc1 and Alp41, we created temperature-sensitive mutants to analyse defective phenotypes, particularly focussing on microtubule structures and dynamics. Interestingly, we have found a range of phenotypes that may be explained by the resulting GTP/GDP state of the G protein. Consistent with Tbc1 being a GAP for Alp41, genetic data supports that Tbc1 negatively regulates Alp41. Biochemical studies have shown that the interactions of these proteins is conserved in fission yeast, and combined with the genetic data we investigated their physiological significance. In addition, we have identified that Arl2/Alp41 also interacts with cofactor D/Alp1 in a GTP/GDP state dependent manner. This led us to an interesting finding that cofactor D/Alp1 possibly has two opposing roles in the cofactor pathway – to assemble and to disassemble microtubules.

Tubulin-specific chaperone D is a microtubule manufacturing chaperone essential to the ability for cells to adopt their appropriate shape, to undergo cell division, as well as to maintain metabolic homeostasis. Recently, homozygous missense mutations to the TBCD gene were reported to cause a severe, early onset neurodegenerative condition in infants. This thesis illuminates the vast and interconnected molecular mechanisms through which TBCD and its clinically-relevant mutations influence cell cycle, metabolism and differentiation.

Das Ziel dieser Arbeit war die Untersuchung des enantiomerenspezifischen Umweltverhaltens des Flammschutzmittels Hexabromcyclododecan (HBCD). Zu Beginn erfolgte daher die Optimierung und Validierung eines enantiomerenspezifischen Analysenverfahrens für die Bestimmung von HBCD in Biota. Die errechneten mittleren Wiederfindungen lagen im Bereich von 100-102 % und die Nachweisgrenzen zwischen 0,131 und 0,255 pg g-1. Untersuchungen zur ubiquitären Verteilung von HBCD erfolgten an Eiern der Silbermöwe deutscher Nord- und Ostseeinseln (Probenahme 1988-2008). In allen Fällen dominierte alpha-HBCD das Diastereomerenmuster, wobei eine bevorzugte Anreicherung von (-)-alpha-HBCD sowie ein zeitlicher Trend aller Enantiomeren-Gehalte festgestellt wurde. Zur Klärung der Frage einer Bioakkumulation sowie -isomerisierung der HBCD-Stereoisomere erfolgten Langzeit-Fütterungsversuche an Spiegelkarpfen. Die Untersuchungen ergaben eine signifikante Akkumulation des jeweils gefütterten HBCD-Enantiomers, jedoch konnte die Hypothese der Bioisomerisierung nicht bestätigt werden. Ein weiterer Schwerpunkt lag in Untersuchungen zur cytochromabhängigen enantiomerenspezifischen Biotransformation von HBCD im Rahmen des Metabolismus an Lebermikrosomen diverser Spezies. Hier konnte gezeigt werden, dass HBCD dem Phase I-Metabolismus unterliegt und hydroxyliert wird. Dabei weist jedes HBCD-Enantiomer ein spezifisches Metabolitenmuster auf, was eine Zuordnung der hydroxylierten Verbindungen zum entsprechenden HBCD-Enantiomer erlaubt. Anhand von Zeitreihen sowie der Berechnung von Halbwertszeiten konnte der Verdacht eines enantiomerenspezifischen Metabolismus in Richtung einer Anreicherung von (-)-alpha- und (+)-gamma-HBCD bestätigt werden. Inkubationsansätze mit reinen Cytochrom (CYP)-Isoformen sowie molekülmechanische Berechnungen legen die Vermutung nahe, dass dem CYP3A4 eine Schlüsselrolle bei der Metabolisierung von HBCD zukommt.
The main emphasis of this thesis was on the enantio-specific environmental behaviour of the polybrominated flame retardant hexabromocyclododecane (HBCD). Initially, an enantio-specific analytical method for the determination of HBCD in biota was optimised and validated. The calculated mean recoveries ranged from 100 to 102 % and the limits of detection are in the range of 0.131 to 0.255 pg g-1. First investigations of the ubiquitous environmental distribution of HBCD were performed using herring gull eggs from different islands in the North and Baltic Sea (sampling 1988 to 2008). In all cases alpha-HBCD was the predominant diastereomer. Significant deviations from the racemic mixture revealed a preferred enrichment of the first eluting (-)-alpha-HBCD. In addition, a temporal trend of HBCD levels was observed. To clarify the issue of accumulation as well as bioisomerisation of HBCD stereoisomers, a long-term feeding study with mirror carps was performed. The results showed an accumulation of each initially fed HBCD enantiomer, but hypothesis of a bioisomerisation could not be confirmed. Another important focus of this work was to study the cytochrome-dependent enantio-specific biotransformation of HBCD enantiomers in various species of liver microsomes. It was shown that HBCD is subject to phase I metabolism. In the course of this process, HBCD is metabolised to hydroxylated products, whereas each HBCD enantiomer results in a specific metabolite pattern allowing the allocation of the corresponding hydroxylated compounds. Investigation of time series as well as the calculation of half-lives, the hypothesis of an enantio-specific metabolism towards an enrichment of (-)-alpha- and (+)-gamma-HBCD could be confirmed. Incubation mixtures with pure cytochrome (CYP) isoforms, as well as molecular mechanic calculations suggest that CYP3A4 plays a key role in the biotransformation processes of HBCD.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
O centrossoma é o principal centro organizador de microtúbulos das células animais, desempenhando funções celulares essenciais no processo de divisão celular, uma vez que regula a nucleação e organização espacial dos microtúbulos, estando também implicado no posicionamento de organelos na célula, como o complexo de Golgi, no estabelecimento da polaridade celular, na migração e adesão celulares e na ciliogénese. Nas células animais o centrossoma encontra-se posicionado no centro da célula em estreita associação com o núcleo. Os cofactores da tubulina (TBCA-E) são proteínas que participam na via de folding da tubulina e possuem funções relacionadas com o citoesqueleto, desempenhando papéis essenciais nas células eucariotas. A proteína TBCCD1 (TBCC-domain containing protein 1) é uma proteína centrossomal relacionada com o TBCC e com a proteína RP2, uma vez que possui os domínios funcionais TBCC e CARP, porém não parece possuir actividade de GAP para a tubulina. O TBCCD1 é um potencial regulador do posicionamento do centrossoma em estreita interacção com o núcleo e da organização citoplasmática. Neste trabalho descrevemos a identificação do domínio responsável pela localização centrossomal da proteína TBCCD1 humana, constituído pelos primeiros 20 resíduos de aminoácidos da sua região N-terminal. Em células humanas observámos que a expressão da proteína TBCCD1 com uma mutação pontual no resíduo de prolina na posição 24 leva à deslocalização da γ-tubulina do centrossoma. Verificámos também que três mutações pontuais distintas nos motivos VxPX e KRAK da proteína causam uma menor eficiência na formação de cílios primários. Concluindo, a proteína centrossomal TBCCD1 humana parece ter uma ligação à γ-tubulina, contudo ainda não está esclarecido se esta interacção é directa ou indirecta, podendo a γ-tubulina ser um parceiro molecular do TBCCD1. O TBCCD1 deverá também ter um papel essencial in vivo resultante do seu envolvimento na manutenção da ligação do centrossoma ao núcleo e no processo de ciliogénese.
The centrosome is the major microtubule organizing center in animal cells, playing an essential role in cellular processes such cell division, since it regulates the nucleation and spatial organization of microtubules, and is also implicated in organelle positioning in the cell, such as the Golgi apparatus, cell polarity establishment, cell migration and adhesion and ciliogenesis. In animal cells, the centrosome is positioned in the center of the cell in close association with the nucleus. The tubulin cofactors (TBCA-E) are proteins involved in tubulin folding pathway that have emerged as proteins with crucial roles in eukaryotic cells related to the cytoskeleton. The TBCCD1 protein (TBCC domain-containing protein 1) is a centrossomal protein related to TBCC and RP2 protein, since it contains the TBCC and CARP domains. However, TBCCD1 probably doesn’t have a GAP activity towards tubulin. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization. In this work we described the identification of the centrosome targeting motif of the human TBCCD1, corresponding to the first 20 amino acid residues of its N-terminus region. Our studies performed in mammalian cell lines revealed that the expression of TBCCD1 with a point mutation in the proline residue at position 24 leads to the mis-localization of γ-tubulin from the centrosome. Furthermore, we also found that three distinct point mutations in the motifs VxPX and KRAK lead to a lower efficiency of transfected cells to assemble primary cilia. Also, the obtained results clearly show that the human centrossomal TBCCD1 protein seems to have an interaction with γ-tubulin, but whether this is direct or indirect is still not clear. However, our results support the idea that γ-tubulin will probably be a molecular partner of TBCCD1. The TBCCD1 should also have an essential role in vivo resulting from their involvement in maintaining the nucleus-centrosome association and its involvement in ciliogenesis.

Des variants pathogéniques du gène TBC1D24 sont associés à des maladies génétiques dont la majorité sont transmises d’une façon autosomique récessive. Les phénotypes sont variables en termes de présentation clinique et de sévérité. Les formes les plus sévères causent une encéphalopathie épileptique (EIEE16) ou le syndrome DOORS qui est marqué par une surdité, des anomalies des ongles et des doigts, un déficit intellectuel et des convulsions qui sont souvent difficiles à contrôler. D’autres formes d’épilepsie incluent EPRPDC (Rolandic epilepsy with paroxysmal exercise-induce dystonia and writer's cramp), FIME (familial infantile myoclonic epilepsy), et PME (progressive myoclonus epilepsy). Une variant faux-sens spécifique est associée à une surdité autosomique dominante (DFNA65) qui se développe à l’âge adulte. Nous avons écrit un guide de pratique clinique qui inclut une revue de la littérature sur les phénotypes publiés chez les individus avec des variantes pathogénique du gène TBC1D24 avec de recommandations pour le suivi clinique de ces patients. De plus, une cohorte de huit patients avec déficience intellectuelle et épilepsie qui partagent une microdélétion sur le chromosome 16p13.3 contenant le gène TBC1D24 a été assemblée et caractérisée afin de définir un nouveau syndrome génétique. La région critique contient TBC1D24, ATP6V0C et PDPK1. Le phénotype similaire entre les huit individus suggère que l’haploinsuffisance pour TBC1D24, ATP6V0C et PDPK1 cause un nouveau syndrome génétique. L’etude des gènes essentiels pour le phénotype dans cette cohorte aide dans l’identification des nouveaux gènes candidates pour la déficience intellectuelle et épilepsie.
Pathogenic variants in the TBC1D24 gene are associated with genetic disorders, the majority of which are transmitted in an autosomal recessive manner. The phenotypes are variable in terms of clinical presentation and severity. The most severe forms cause epileptic encephalopathy (EIEE16) or DOORS syndrome which is marked by deafness, abnormalities of the nails and fingers, intellectual deficit and convulsions which are often difficult to control. Other forms of epilepsy include EPRPDC (Rolandic epilepsy with paroxysmal exercise-induce dystonia and writer's cramp), FIME (familial infantile myoclonic epilepsy), and PME (progressive myoclonus epilepsy). A specific missense variant is associated with autosomal dominant deafness (DFNA65) which develops in adulthood. A review of the literature of the published phenotypes observed in individuals with pathogenic variants in the TBC1D24 gene is presented here with recommendations for the clinical management of these patients. In addition, a group of eight patients with intellectual disability and epilepsy who share a microdeletion on chromosome 1613.3 containing the TBC1D24 gene were characterized in order to define a new genetic syndrome. The critical region contains TBC1D24, ATP6V0C and PDPK1. The significantly similar phenotype shared by the eight individuals suggests that haploinsufficiency for TBC1D24, ATP6V0C and PDPK1 causes a new genetic syndrome. Knowledge of the genes essential for the phenotype in this cohort helps in the identification of new candidate genes for intellectual disability and epilepsy.

碩士
中華醫事科技大學
醫學檢驗生物技術系碩士班
100
In this study,we use both Immuno-chromatography (ICA) assay,which detects MPB-64 antigen of TB,and Nucleic acid amplification test,which detects IS 6110 gene of TB.The samples were processed with tuberculisos culture from MGIT,and checking cord formation by Ziehl-Neelsen stain,sequentially.The result shows that the time to identify takes 6.9,9.2,10.6,13.8,11.6,17.1 days while the smear of sputum was4+,3+,2+,1+,scanty and negative,respectively. The sensitivity, specificity, false negatives (FN), false positives (FP), positive predictive value (PPV), negative predictive value (NPV), and accuracy of Immuno-chromatography assay were 91.9%, 98.2%, 8.1%, 1.8%, 96.6%, 95.7%, and 96.0%, respectively. On the other hand, Nucleic acid amplification test was 95.9%, 91.1%, 4.0%, 8.8%, 85.6%, 97.6%, and 92.9%, respectively. According the results, the NAA had more false positives than ICA (8.8% v.s. 1.8%, p<0.05), consequently ICA showed higher specificity and positive predictive value than NAA (98.2%, 96.6% v.s. 91.1%, 85.6%; p<0.05).Above all results got significant difference (p<0.05), but sensitivity, FN, NPV, and accuracy didn’t (91.9%, 8.1%, 95.7%, 96.0% v.s. 95.9%, 4.0%, 97.6%, 92.9%, p>0.05). Therefore, Immuno-chromatography assay (ICA) will be an excellent tool for MTB identification because of its rapidity, easiness, device-free, and lower cost. In strong smear-positive case, the identification results will be reported about 1 week by MGIT culture, checking cord formation, and ICA test. In conclusion, ICA could be a good choice other than NAA to provide tuberculosis diagnosis and identification.

Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2019
O centrossoma é o principal centro organizador de microtúbulos nas células animais. Este desempenha diversas funções celulares como a organização espacial do citoplasma e dos organelos, bem como na formação do fuso mitótico durante a divisão, na migração celular, no estabelecimento do eixo de polaridade intrínseco da célula “Núcleo-Centrossoma-Complexo de Golgi” e na ciliogénese. O nosso grupo tem-se focado no estudo da proteína centrossomal TBCCD1 (TBCC domain-containing protein 1) e nas funções que esta desempenha. Nos seus primeiros estudos, o nosso grupo desvendou um fenótipo característico quando a proteína TBCCD1 era depletada (Gonçalves, 2010). O mesmo determinou recentemente que a proteína TBCCD1 estabelecia uma vasta rede de interações com outras proteínas, após a realização de BioID (Camelo, 2015). Como tal, identificaram-se cerca de 82 proteínas pertencentes a este interatoma da TBCCD1. Nestas 82 proteínas, destacou-se um grupo de seis proteínas, devido a sua capacidade de estabelecerem interações entre si, bem como com as restantes proteínas deste interatoma da TBCCD1 (dados ainda não publicados). As proteínas reunidas nesse grupo são essencialmente proteínas codificadas por genes envolvidos em ciliopatias, isto é, doenças associadas a defeitos relacionados com a biogénese de cílios primários. Uma das proteínas associadas a ciliopatias encontrada neste grupo foi a proteína OFD1 (Oral-Facial-Digital-1). Esta proteína localiza-se principalmente na região distal do centríolo-mãe e nos satélites centriolares. A proteína é traduzida pelo gene ofd1 localizado no cromossoma X. Mutações deste gene levam à manifestação da síndrome Oral-Facial-Digital, que se caracteriza por anormalidade orais, dimorfismo facial e defeitos no sistema nervoso central (SNC) tais como microcefalias (Lopes, et al., 2011). Neste trabalho estudámos as interações entre a proteína TBCCD1 e a proteína OFD1 e a influência desta interação no posicionamento do centrossoma, e nos satélites centriolares. Diversos ensaios de depleção ou de sobre-expressão da proteína TBCCD1 foram realizados e, seguidamente, observados por imunofluorescência e immunoblotting, de modo a estudar o impacto que diferentes níveis fisiológicos da TBCCD1 teriam na proteína OFD1. Com este trabalho foi possível concluir que as proteínas TBCCD1 e OFD1 estabelecem algum tipo de relação, dado que, tanto nos ensaios de depleção ou de sobre-expressão de TBCCD1 se verifica uma diminuição da proteína OFD1. Também se concluiu que parece existir um intervalo de níveis fisiológicos da proteína TBCCD1 que, sendo ultrapassados, têm uma influência direta na proteína OFD1, provocando uma diminuição da mesma na célula. Por fim, foi ainda possível estabelecer a hipótese de que a proteína TBCCD1 estabelece uma interação com os complexos proteicos constituídos com a proteína OFD1 (Chevrier, et al., 2015), ou ainda que, possivelmente, a própria proteína TBCCD1 se associa a um dos complexos formados pela proteína OFD1.
The centrosome is the main organizing center for microtubules in animal cells. It performs several cellular functions such as the spatial organization of the cytoplasm and organelles, as well as the formation of the mitotic spindle during division, cell migration, establishment of the intrinsic cell polarity axis "Nucleus-Centrosome-Golgi Complex" and in ciliogenesis. Our group has been focused on studying the centrosomal protein TBCCD1 (TBCC domain-containing human protein 1) and its functions. In our early studies, the group uncovered a characteristic phenotype when the TBCCD1 protein was depleted (Gonçalves, 2010). It was recently determined through BioID analysis that the TBCCD1 protein established a vast network of interactions with other proteins (Camelo, 2015). As such, about 82 proteins belonging to this TBCCD1 interatom were identified. Out of those 82 proteins, a group of six proteins was highlighted due to their ability to establish interactions among themselves, as well as with the remaining proteins of this TBCCD1 interatoma (data yet not published). The proteins included in this group are essentially proteins encoded by genes involved in ciliopathies, i.e., diseases associated with defects related to the biogenesis of primary cilia. One of these ciliopathy-associated proteins found in the referred group of interest was OFD1 (Oral-Facial-Digital-1). This protein is mainly located in the distal region of the mother centriole and centriolar satellites. The protein is transduced by the gene ofd1 located on the X chromosome. Mutations of this gene lead to the manifestation of the Oral-Facial-Digital syndrome, which is characterized by oral abnormalities, facial dimorphism and central nervous system (CNS) defects such as microcephaly (Lopes, et al., 2011). In this work we studied the interactions between the TBCCD1 protein and the OFD1 protein and the influence of this interaction on centrosome position and on the centriolar satellites. Several assays of depletion or of overexpression of the TBCCD1 protein were performed and then observed by immunofluorescence and immunoblotting, in order to study the impact that different physiological levels of TBCCD1 would have on the OFD1 protein. This work allowed us to conclude that the TBCCD1 and OFD1 proteins establish some type of relationship, since in both TBCCD1 depletion or overexpression assays occurs a decrease of OFD1 protein. It was also concluded that there appears to be a range of physiological levels of the protein TBCCD1, which if overtaken has a direct influence on the OFD1 protein, causing a decrease of the later in the cell. At last, it was also formulated the hypothesis that the TBCCD1 protein interacted with protein complexes constituted with OFD1 protein (Chevrier, et al., 2015), or even possibly the TBCCD1 protein itself associates with one of the complexes formed by the OFD1 protein.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
A proteína humana centrossomal TBCCD1 está envolvida na ligação do centrossoma ao núcleo. A diminuição dos níveis do TBCCD1 causa a quebra do eixo de polaridade intrínseca “Núcleo-Centrossoma-Complexo de Golgi” em consequência da anormal localização do centrossoma na periferia da célula a qual é acompanhada por uma desorganização do complexo de Golgi. Recentemente, foram identificadas variantes de splicing alternativo desta proteína que se localizam exclusivamente no citoplasma. Neste trabalho pretendeu-se esclarecer as funções da proteína TBCCD1 e da TBCCD1 variante 2, através i) do estudo do efeito da depleção da TBCCD1 na dinâmica dos microtúbulos e no citoesqueleto de actina e ii) da identificação das suas proteínas interatuantes, recorrendo ao método de BioID. Apesar de a TBCCD1 ter um domínio que a associa com a actina e interagir com várias proteínas que têm como funções polimerizar os filamentos de actina, em particular nas junções célula-célula e de adesão, não foi possível identificar nenhuma relação clara entre a TBCCD1 e os filamentos de actina nas células depletadas de TBCCD1. Este trabalho permitiu concluir que a TBCCD1 tem um papel importante na estabilização dos microtúbulos em células hTERT-RPE-1, em particular de uma subpopulação de microtúbulos que rodeia o núcleo e que se localiza preferencialmente do lado do centrossoma. Esta subpopulação de microtúbulos regula parcialmente o posicionamento do centrossoma em relação ao núcleo nestas células. Observou-se ainda que a depleção da TBCCD1 causa a dispersão da proteína centrossomal CEP170, que se associa a esta subpopulação de microtúbulos, e fica dispersa no citoplasma das células depletadas de TBCCD1. Neste trabalho foi também possível estabelecer o interatoma da TBCCD1 e da TBCCD1 variante 2. Assim, observou-se que a TBCCD1 interatua com proteínas com distintas funções celulares, estando principalmente envolvida na dinâmica dos microtúbulos, com a integridade do centrossoma e na polaridade celular, particularmente no estabelecimento e manutenção das junções celulares. A análise do interatoma, conjuntamente com os resultados obtidos no estudo da dinâmica dos microtúbulos, sugere que a TBCCD1 seja uma proteína adaptadora que promove a interação entre diferentes proteínas. A TBCCD1 variante 2, por outro lado, interage sobretudo com chaperones moleculares, proteínas mitocondriais e centroméricas. Embora à primeira vista estas interações possam parecer não estar relacionadas, uma análise mais detalhada mostra que a TBCCD1 variante 2 poderá estar envolvida na montagem e organização de estruturas complexas como os cinetocoros e a organização do genoma mitocondrial através da interação direta com os diferentes componentes ou através da interação com chaperones moleculares.
The human centrosomal protein TBCCD is involved in the connection between the centrosome and the nucleus. Lower TBCCD1 levels lead to a disrupture of the intrinsic cell polarity axis “Nucleus-Centrosome-Golgi Apparatus” due to an abnormal localization of the centrosome in the cell periphery, which is accompanied by the fragmentation of the Golgi apparatus. Recently, TBCCD1 splicing variants that localize exclusively in the cytoplasm have been identified. In this work we aimed at clarifying what are the cellular functions of TBCCD1 and TBCCD1 variant 2 by i) studying the effect of TBCCD1 depletion in the microtubule network and actin cytoskeletons dynamics and ii) identifying their interacting proteins using the BioID method. Although TBCCD1 has a domain able to associate this protein with actin and TBCCD1 interacts with several proteins that have a role in actin polymerization, particularly in cell-cell and adhesion junctions, it was not possible to identify a clear relationship between TBCCD1 and the actin filaments in TBCCD1-depleted cells. This work allowed us to conclude that TBCCD1 has an important role in microtubule stabilization in hTERT-RPE-1 cells, in particularly in a microtubule subpopulation surrounding the nucleus which is localized preferentially near the centrosome. This microtubule subpopulation partially regulates the centrosome-nucleus positioning. We also observed that TBCCD1 depletion causes cytoplasmic dispersion of the centrosomal protein CEP170, which is associated with this microtubule subpopulation. In this work we also established the TBCCD1 and TBCCD1 variant 2 interactomes. TBCCD1 interacts with several proteins with distinct functions, mainly involved in microtubule dynamics, centrosome integrity and cell polarity, and also in the establishment and maintenance of cell junctions. Both the interactome analysis and the results obtained with the microtubule dynamic studies suggest that TBCCD1 may function as a promoter of the interaction between distinct proteins. TBCCD1 variant 2, in other hand, interacts mainly with molecular chaperones and mitochondrial and centromeric proteins. Although it may seem that these interactions are not related with which other, a more detailed analysis shows that TBCCD1 variant 2 protein can be involved in the assembly and organization of complex structures, such as the kinetochores and the organization of the mitochondrial genome either through the direct interaction with the different components or through the interaction with molecular chaperones.

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2010.
O centrossoma é o principal organizador de microtúbulos das células animais e desempenha funções celulares fundamentais estando implicado por exemplo, na organização espacial do citoplasma, no posicionamento de organelos como o complexo de Golgi, na formação do fuso mitótico, no estabelecimento da polaridade celular, na migração celular e na ciliogénese. O centrossoma encontra-se activamente associado com o núcleo na célula em interfase. Contudo, os mecanismos subjacentes a este posicionamento não se encontram totalmente esclarecidos. Os cofactores da tubulina (TBCA-E), proteínas que participam na via de “folding” da tubulina, têm vindo a ser descritos como tendo papéis cruciais na célula, nem sempre directamente relacionados com o “folding”. Um dos cofactores menos estudado, o TBCC, actua como uma GAP (de “GTPase activating protein”) para a -tubulina. A proteína RP2 (de “retinitis pigmentosa protein”), relacionada com o TBCC nos seus domínios funcionais, também actua como GAP para a -tubulina e para a proteína Arl3 (de “ADP-ribosylation factor-like 3”). Neste trabalho descrevemos a primeira caracterização da proteína humana TBCCD1 (de “TBCC-domain containing protein”), que possui os domínios TBCC e CARP, sendo portanto relacionada com o TBCC e a proteína RP2. Porém, o TBCCD1 não parece possuir actividade de GAP para a tubulina, uma vez que um ensaio de complementação em Saccharomyces cerevisae, mostrou que o TBCCD1 é incapaz de reverter os fenótipos da delecção do gene cin2 (homólogo do tbcc). Em células humanas observámos que o TBCCD1 se localiza no centrossoma, na zona mediana do fuso mitótico, no “midbody” e no corpo basal de cílios primários e motores. Com o silenciamento do tbccd1 em células RPE-1 observou-se: i) um aumento do tamanho das células e um atraso/paragem do ciclo celular em G1; ii) um afastamento do centrossoma em relação ao núcleo; iii) a desorganização do complexo de Golgi; iv) uma diminuição da eficiência de produção de cílios primários; v) uma migração celular dirigida mais lenta. Concluindo, a proteína TBCCD1 humana deverá ter um papel crucial in vivo resultante do seu papel na manutenção da associação do centrossoma ao núcleo o que afecta a divisão celular, a organização do Golgi, a ciliogénese e a migração celular. Esta hipótese é suportada por resultados preliminares obtidos com o silenciamento do gene tbccd1 em embriões de peixe zebra que resulta em problemas no desenvolvimento embrionário como a curvatura dos embriões e edema cardíaco. Curiosamente, estes fenótipos têm vindo a ser correlacionados com problemas ciliares.
Fundação para a Ciência e a Tecnologia (FCT)-bolsa de Doutoramento (SFRH/BD/24532/2005); Instituto Gulbenkian de Ciência (IGC)

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz
Nos últimos anos têm sido realizados inúmeros estudos sobre espécies reactivas de oxigénio (EROs), devido ao seu envolvimento em processos como o cancro e envelhecimento. Contudo, as EROs são subprodutos normais do metabolismo celular, podendo também ter origem em fontes exógenas à célula. As ERO, como o peróxido de hidrogénio (H2O2), são espécies muito instáveis e quimicamente bastante reactivas, com capacidade de causar vários danos graves nas diferentes biomoléculas. O H2O2 é in vivo um oxidante suave contudo, devido à sua permeabilidade através das membranas celulares tem sido alvo de grande interesse, estando implicado em processos de desencadeamento e modulação de vias de sinalização celulares. O interesse de estudar o H2O2 como um possível regulador de algumas proteínas centrossomais surgiu através de um estudo retrospectivo, em que foi verificada a associação entre altos níveis de H2O2, bolus addiction, e a hiperamplificação dos centrossomas e formação de fusos mitóticos multipolares. Posto isto, como consequência foi verificado um aumento de células multinucleadas, este que por sua vez está associado a vários processos patológicos. Face a estes resultados, propôs-se como hipótese que o H2O2 em baixas concentrações, semelhantes às encontradas fisiologicamente, poderia modelar os níveis de algumas proteínas centrossomais, como o TBCCD1 (de TBCC-domain containing protein 1), a γ-tubulina e a pericentrina. O TBCCD1 é um potencial regulador do posicionamento do centrossoma e por consequência da organização interna da célula. A γ-tubulina e a pericentrina desempenham funções específicas ao nível da nucleação dos microtúbulos. Neste estudo, foram usadas duas linhagens celulares (hTERT- RPE-1 e HeLa) as quais foram expostas, através do método de titulação de H2O2 em estado estacionário a uma concentração constante de 10 e 25μM de H2O2 , respectivamente, durante um determinado período de tempo. Os níveis das proteínas em estudo foram analisados por Western blotting. Como resultados, não foram identificadas alterações significativas nos níveis da proteína TBCCD1, em ambas as linhagens, e na γ-tubulina em h-TERT-RPE-1. No caso da pericentrina (hTERT- RPE-1 e HeLa), foi observada uma diminuição dos níveis proteicos, podendo estar relacionado com uma diminuição da capacidade nucleadora do centrossoma, uma vez que a pericentrina recruta os complexos γ- TuRC para esta estrutura.

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2010
O peróxido de hidrogénio (H2O2) embora seja um oxidante relativamente fraco, é capaz de alterar o nível de oxidação dos grupos tióis ao reagir com resíduos de cisteínas. Essas propriedades tornam o H2O2 uma das espécies reactivas de oxigénio (ERO) com potencialidade para participar em vias de sinalização. Deste modo, o desencadeamento de uma determinada via de sinalização, ou a sua modulação, deverá ocorrer pelo aumento temporário das concentrações de H2O2, como por exemplo, durante a resposta inflamatória. Por outro lado, a exposição prolongada a algumas ERO têm vindo a ser relacionada com o início, progressão e desenvolvimento tumoral. Muitos dos tumores são caracterizados por aneuploidia e anormalidades centrossomais. Por exemplo, células de mamífero expostas a H2O2, por bolus addition, apresentam centrossomas supra-numerários. Neste contexto iniciou-se o estudo da influência de concentrações sinalizadoras de H2O2 nos níveis de duas proteínas centrossomais, a proteína TBCCD1 (TBCC-domain containing protein 1) e a y−tubulina nas linhas celulares humanas HeLa e hTERT-RPE-1. O TBCCD1 é um potencial regulador do posicionamento do centrossoma e da organização citoplasmática, a y−tubulina é uma proteína chave na nucleação dos microtúbulos nos centros organizadores de microtúbulos, como o centrossoma. Por western blot mostrou-se, em ambas as linhas celulares, o aumento dos níveis de TBCCD1 ao longo do tempo de exposição de H2O2 em estado estacionário. A análise da velocidade de consumo de H2O2 em células hTERT-RPE-1 que sobre-expressam a proteína de fusão TBCCD1-GFP mostrou um relação directa desta com o aumento dos níveis de TBCCD1-GFP. Esta proteína, na presença de H2O2, varia de modo análogo à proteína endógena. Estudos similares mostraram a diminuição dos níveis de y−tubulina ao longo do tempo de exposição de H2O2. Por microscopia de imunofluorescência indirecta observou-se também a diminuição dos níveis desta proteína no centrossoma em células HeLa. Observou-se ainda uma alteração da capacidade de nucleação dos microtúbulos pelo centrossoma e uma modificação na organização destes após repolimerização em células previamente tratadas com nocodazole e após remoção desta. No conjunto, estes resultados mostram que ambos os níveis destas proteínas variam na presença de baixos concentrações de H2O2, constituindo este trabalho uma abordagem inicial do efeito do H2O2 em concentrações reguladoras no centrossoma.
The hydrogen peroxide (H2O2) although a relatively weak oxidant, is highly reactive with sulfhydryl groups. These properties make the H2O2 a reactive oxygen species (ROS) with the potential to participate as a regulator in signaling pathways. Thus, the temporary increase in the concentrations of H2O2 should trigger or modulate a signal pathway, such as during the inflammatory response. Moreover, prolonged exposure to some ROS has been related with the initiation, progression and tumor development. Many tumors are characterized by aneuploidy and centrosomal abnormalities. For example, mammalian cells exposed to H2O2 by bolus addition showed supra-numerary centrosomes. In this context we began the study of the influence of signaling concentrations of H2O2 in the levels of two centrosomal proteins, protein TBCCD1 (TBCC domain-containing protein 1) and y − tubulin in HeLa and hTERT-RPE-1 human cell lines. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization and the y − tubulin is a key protein in the nucleation of microtubules on microtubule organizing centers, such as the centrosome. By western blot, we detected in both cell lines, increased levels of TBCCD1 over time of exposure to a steady state of H2O2. The analysis of H2O2 consumption by intact hTERT-RPE-1 cells that over-expresses the fusion protein TBCCD1-GFP showed a direct relationship of this with increased levels of TBCCD1-GFP. Noteworthy, in the presence of H2O2, the TBCCD1-GFP levels, showed the same variation that those of the endogenous protein. Similar studies showed that −tubulin levels decreased of upon an exposition to H2O2. By immunolocalization microscopy we observed in HeLa cells a decrease of y − tubulin’s levels at the centrosome. To investigate if this observation has an impact in the ability of centrosomes to nucleate microtubules we have treated HeLa cells, previously exposed to steady-state levels of H2O2, with the antimitotic drug nocodozole. After washing out this microtubule depolymerizing agent we follow microtubule repolymerization by immunofluorescence microscopy. We observed that cells treated with H2O2 present a different ability to nucleate microtubules in comparison to control cells. At end these cells presented a distinct organization of the microtubule cytoskeleton. Taken together the results presented in this work show that a low concentration of H2O2 promotes a variation of the TBCCD1 and y − tubulin’s levels. Therefore, this work constitutes an initial approach to the study of the impact at the centrosome of H2O2 at levels expected for a signaling role.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
Com a identificação e o estudo do gene humano tbccd1, foi identificada uma proteína, a TBCCD1, por ele codificada. Esta proteína foi então descrita como sendo uma proteína centrossomal, que se localiza também nos corpos basais de cílios primários e no corpo médio. Foram identificados dois novos transcritos do tbccd1 resultantes de splicing alternativo, nomeadamente, variante 2 e variante 3, sendo que o transcrito variante 1 codifica a proteína TBCCD1 variante 1 inicialmente descrita. O knockdown do tbccd1 utilizando siRNAs provoca vários fenótipos em células hTERT RPE-1, como o aumento da distância centrossoma-núcleo, a fragmentação do complexo de Golgi e a diminuição na velocidade de migração em ensaios de fecho da ferida. Assim, este trabalho teve como principal objetivo o estudo da função biológica de cada uma das variantes identificadas. Estudou-se a localização celular de cada uma das variantes identificadas em diferentes linhas celulares humanas. Observou-se que as proteínas TBCCD1 variante 2 e TBCCD1 variante 3 têm uma localização citoplasmática em todas as situações analisadas. Pelo contrário, como tinha já sido observado, a proteína TBCCD1 variante 1 localiza-se no centrossoma, no corpo médio e nos corpos basais de cílios primários. Curiosamente, observou-se que a localização desta proteína no corpo médio é dependente dos microtúbulos, ao contrário do que acontece com a sua localização centrossomal. Esta observação sugere então uma interação com microtúbulos do corpo médio onde existe uma acumulação de microtúbulos acetilados. Observou-se também que a sobre expressão das proteínas TBCCD1 variante 1 ou TBCCD1 variante 2 provoca uma diminuição nos níveis de α-tubulina acetilada, assim como de γ-tubulina e de β-actina. Já a sobre expressão da proteína TBCCD1 variante 3 não parece afetar os níveis dos componentes do citoesqueleto analisados. Estudou-se também se o fenótipo do aumento da distância centrossoma-núcleo pode ser provocado por apenas uma das variantes ou resulta da ação combinada de mais do que uma variante. Para isso, realizaram-se ensaios de recuperação do fenótipo, tendo-se observado que as proteínas TBCCD1 variante 1 e TBCCD1 variante 2 revertem parcialmente o fenótipo em estudo. Para além disso, realizou-se um estudo recorrendo a RT-qPCR no qual se observou que as variantes têm uma função de regulação entre si. A sobre expressão das proteínas TBCCD1 variante 1 ou TBCCD1 variante 2 leva À alteração dos níveis dos transcritos das variantes 1, 2 e 3. Já a sobre expressão da proteína TBCCD1 variante 3 afeta apenas os níveis do transcrito variante 3 endógeno. Em conjunto, os resultados obtidos neste estudo indicam que as proteínas codificadas pelos três transcritos identificados deverão ter diferentes funções celulares. Para além disso, as proteínas TBCCD1 variante 1 e TBCCD1 variante 2 afetam os níveis de α-tubulina acetilada, γ-tubulina e β-actina, o que pode afetar, por exemplo, a organização do complexo de Golgi e a migração celular, através da regulação da dinâmica dos microtúbulos. Assim, estes resultados contribuem de forma decisiva para a compreensão dos fenótipos observados nas experiências do knockdown do tbccd1.
When the human gene tbccd1 was identified and studied, a protein, TBCCD1, that it encoded was also identified. This protein was then described as a centrossomal protein that also localizes at the basal bodies of primary cilia and to the midbody. Two new transcripts of tbccd1 originated by alternative splicing were identified, namely, variant 2 and variant 3, and it is now known that variant 1 encodes the TBCCD1 protein initially described. The knockdown of tbccd1 using siRNAs causes several phenotypes in hTERT RPE-1 cells, such as the increase in centrosome-nucleus distance, fragmentation of the Golgi apparatus and a decrease of the cell migration in wound healing assays. The main goal of this work was to study the biological function of each of the identified tbccd1 variants. In this study, the cellular localization of each of the variants in different human cell lines was accessed. TBCCD1 variant 2 and TBCCD1 variant 3 proteins have a cytoplasmic localization in all the situations analyzed. As it has already been described, TBCCD1 variant 1 protein localizes at the centrosome, the basal bodies of primary cilia and to the midbody. Interestingly, it was shown that the midbody localization of this protein is dependent on microtubules, while its centrossomal localization is not. This observation suggested an interaction of this protein with a population of microtubules in the midbody where they are highly acetylated. Overexpression of TBCCD1 variant 1 or TBCCD1 variant 2 causes a decrease on acetylated α-tubulin levels, and also on γ-tubulin and β-actin levels. The overexpression of TBCCD1 variant 3 does not seem to affect the levels of any of the cytoskeleton components analyzed. It was also studied if the increased centrosome. Nucleus distance phenotype is caused by only one or a combination of the identified variants. In order to do this, we performed phenotype rescue assays, and it was shown that overexpression of TBCCD1 variant 1 or TBCCD1 variant 2 can partially rescue this phenotype. Moreover, we used RT-qPCR to study whether the identified variants have a regulatory function between them, and showed that TBCCD1 variant 1 or TBCCD1 variant 2 overexpression affects the levels of the three transcripts. On the other hand, the overexpression of TBCCD1 variant 3 only affects variant 3 endogenous transcript levels. Taken together, the results obtained in this study indicate that the proteins encoded by the three transcripts might have different cellular functions. Furthermore, TBCCD1 variant 1 and TBCCD1 variant 2 affect acetylated α-tubulin, γ-tubulin and β-actin levels which may be implicated on, for example, Golgi apparatus organization and cell migration by the regulation of microtubules dynamics. Therefore, this results contributed to the comprehension of the causes of the tbccd1 knockdown phenotypes.

La déficience intellectuelle (DI) définit un groupe de conditions génétiquement hétérogènes caractérisées par l’apparition de troubles cognitifs précoces chez l’enfant. Elle affecte 1-3% de la population dans les pays industrialisés. La prévalence de la DI est beaucoup plus élevée ailleurs dans le monde, en raison de facteurs sociodémographiques comme le manque de ressources dans le système de santé, la pauvreté et la consanguinité. Des facteurs non-génétiques sont mis en cause dans l’étiologie de la DI ; on estime qu’environ 25% des cas de DI sont d’origine génétique. Traditionnellement, les bases moléculaires de la DI ont été investiguées par des analyses cytogénétiques, les approches de cartographie génétique et le séquençage de gènes candidats ; ces techniques de génétiques classiques sont encore mises à rude épreuve dans l’analyse de maladies complexes comme la DI. La DI liée à l’X a été particulièrement étudiée, avec plus d’une centaine de gènes identifiés uniquement sur le chromosome X. Des mutations hétérozygotes composites sont mises en évidence dans la DI autosomique, dans le contexte d’unions non-consanguines. L’occurrence de ce type de mutations est rare, chez des individus non-apparentés, de sorte que les mutations dominantes de novo sont plus courantes. Des mutations homozygotes sont attendues dans les populations consanguines ou marquées par un effet fondateur. En fait, les bases moléculaires de la DI autosomique ont été presqu’exclusivement étudiées dans le contexte de populations avec des forts taux de consanguinité. L’origine de la DI demeure encore inconnue dans environ 60 % des cas diagnostiqués. En l’absence de facteurs environnementaux associés à la DI chez ces individus, il est possible d’envisager que des facteurs génétiques non identifiés entrent en jeu dans ces cas de DI inexpliqués. Dans ce projet de recherche, nous voulions explorer l’origine génétique de la DI, dans vingt familles, où une transmission de la maladie selon un mode autosomique récessif est suspectée. Nous avons mis de l’avant les techniques de séquençage de nouvelle génération, afin de mettre en évidence les déterminants génétiques de la DI, à l’échelle du génome humain. En fait, nous avons priorisé la capture et le séquençage de l’exome; soient la totalité des régions codantes du génome humain et leurs sites d’épissage flanquants. Dans nos analyses, nous avons ciblé les variants qui ne sont pas rapportés trop fréquemment dans différentes bases de données d’individus contrôles, ces mutations rares cadrent mieux avec une condition comme la DI. Nous avons porté une attention particulière aux mutations autosomiques récessives (homozygotes et hétérozygotes composites) ; nous avons confirmé que ces mutations ségréguent avec une transmission récessive dans la famille à l’étude. Nous avons identifié des mutations dans des gènes pouvant être à l’origine de la DI, dans certaines des familles analysées ; nous avons validé biologiquement l'impact fonctionnel des mutations dans ces gènes candidats, afin de confirmer leur implication dans la pathophysiologie de la DI. Nous avons élucidé les bases moléculaires de la DI dans huit des familles analysées. Nous avons identifié le second cas de patients avec syndrome de cassure chromosomique de Varsovie, caractérisé par des dysfonctions de l’ARN hélicase DDX11. Nous avons montré qu’une perte de l’activité de TBC1D7, une des sous-unités régulatrice du complexe TSC1-TSC2, est à l’origine de la pathologie dans une famille avec DI et mégalencéphalie. Nous avons mis en évidence des mutations pathogéniques dans le gène ASNS, codant pour l’Asparagine synthétase, chez des patients présentant une microcéphalie congénitale et une forme progressive d’encéphalopathie. Nous avons montré que des dysfonctions dans la protéine mitochondriale MAGMAS sont mises en cause dans une condition caractérisée par un retard prononcé dans le développement associé à une forme sévère de dysplasie squelettique. Nous avons identifié une mutation tronquant dans SPTBN2, codant pour la protéine spinocerebellar ataxia 5, dans une famille avec DI et ataxie cérébelleuse. Nous avons également mis en évidence une mutation dans PIGN, un gène impliqué dans la voie de biosynthèse des ancres de glycosylphosphatidylinositol , pouvant être à l’origine de la maladie chez des individus avec épilepsie et hypotonie. Par ailleurs, nous avons identifié une mutation - perte de fonction dans CLPB, codant pour une protéine chaperonne mitochondriale, dans une famille avec encéphalopathie néonatale, hyperekplexie et acidurie 3-méthylglutaconique. Le potentiel diagnostic des techniques de séquençage de nouvelle génération est indéniable ; ces technologies vont révolutionner l’univers de la génétique moléculaire, en permettant d’explorer les bases génétiques des maladies complexes comme la DI.
Intellectual disability (ID) regroups greatly heterogeneous conditions that are characterized by early-onset cognitive impairment. ID affects about 1-3% of Western populations; but its prevalence is much higher in deprived regions of the world where socio-demographic factors like poor healthcare, lack of resources and parental consanguinity prevail. Non-genetic factors are involved in the etiology of ID; approximately 25% of ID cases are of genetic origin. Traditionally, the molecular basis of ID have been assessed through cytogenetic analyses, genetic mapping and candidate gene approaches. These classical genetic tools are still put to the test in the study of complex diseases like ID. Until recently, X-linked ID cases were the main focus of studies on ID with more than hundred ID genes identified only on the X chromosome. Compound heterozygous mutations are identified in autosomal forms of ID, in the context of non-consanguineous unions. However, the occurrence of such mutations is rare in outbred populations, so that dominant de novo mutations are most common in unrelated individuals. Homozygous mutations are expected in consanguineous unions or in populations marked by a founder effect. In fact, the molecular bases of autosomic recessive ID have been almost exclusively studied in populations with high consanguinity rates. ID remains unsolved in more than 60% of patients. In the absence of environmental factors associated with ID in these individuals, it is possible to consider that unidentified genetic factors are involved in these unexplained ID cases. In this research project, we used next generation sequencing technologies to highlight the genetic causes of ID in twenty families were an autosomal recessive mode of inheritance is expected. We prioritized the use of whole-exome sequencing, namely all coding exons in the genome of this individual. In our analyses, we filtered out variants that were too common in control individuals to describe a rare condition like ID. We focussed our attention on rare autosomic recessive varaiants (homozygous and compound heterozygous), these mutations were confirmed by Sanger re-sequencing to segregate with an autosomal recessive mode of inheritance in the family. We identified mutations in candidate genes for ID in some of the family analysed, we validated the functional impact of the mutations in these genes to confirm their involvement in the pathophysiology of ID in the family studied. We explained the molecular basis of ID in eight of the families studied. We identified the second case of Warsaw-Breakage-Syndrome, a rare genetic disorder characterised by dysfunction of the RNA helicase DDX11. We showed that disruption in TBC1D7, a functional subunit of the TSC1-TSC2 protein complex, cause ID and megalencephaly. We demonstrated that ASNS, the Asparagine Synthetase gene, is defective in patients with congenital microcephaly and progressive encephalopathy. We showed that the gene coding for the mitochondrial protein MAGMAS is involved in the pathophysiology of a condition characterised by developmental delay and severe skeletal dysplasia. We identified a truncating mutation in SPTBN2, encoding for the spinocerebellar ataxia 5 proteins, in a family with ID and spinocerellar ataxia. We also identified a mutation in a gene involved in the biosynthetic pathway of glycosylphosphatidylinositol anchors; the mutation in PIGN may cause the epilepsy and hypotonia features observed in the affected individuals of that family. Finally, we identified a loss of function mutation in CLPB, coding for a mitochondrial chaperone, in individuals with severe encephalopathy, hypereklexia and 3-methylglutaconic aciduria. The diagnostic potential of next generation sequencing technologies is undeniable. These technologies will revolutionize the world of molecular genetics; they will help deciphering the molecular basis of complex diseases like ID.

Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2019
Although poorly studied so far, the centrosomal protein KIAA0753, also known as Moonraker or OFIP, has been implied in the recruitment of other proteins to the centrosome, being involved in various cellular functions, such as centriolar duplication and microtubule stabilization. Moreover, there are mutated versions of the gene that codifies for this protein associated with a group of rare diseases called ciliopathies. Ciliopathies are typified by clinical manifestations that often overlap, such as infertility, obesity, problems regarding cerebral and skeleton development, blindness and polycystic kidneys. Thus, it is assumed that KIAA0753 also has a role in ciliogenesis. In this work, we aimed at clarifying the role of KIAA0753 in the centrosome, by studying the effect of KIAA0753 depletion both in centrosome and in the dynamics of the microtubule cytoskeleton. Our study allowed us to conclude that KIAA0753 may be involved in the maintenance of the centrosome structure and, consequently, in the organization of the microtubule skeleton. Moreover, it was also possible to show that KIAA0753 may, in fact, be involved in the assembly and regulation of cilia length. The protein KIAA0753, as well as other proteins encoded by genes associated with ciliopathies, was identified in work done by our research group as belonging to the interactome of the human centrosomal protein containing the TBCC domain (TBCCD1), which is involved in the correct positioning of the centrosome and in the biogenesis of cilia. Thus, in a parallel study, we also aimed to study whether TBCCD1 interacts with KIAA0753 because TBCCD1 is also a component of the ternary complex that KIAA0753 is part of, or whether this interaction involves an indirect mechanism. Although it was not possible to obtain any evidence for TBCCD1 being one of the constituents of the complex, it was possible to conclude that a functional relationship between the two proteins exists, and that TBCCD1 may have a role in the recruitment of KIAA0753 to the centrosome, which supports the hypothesis of tbccd1 being a new ciliopathic gene.

Although Chlamydomonas reinhardtii is a widely used model organism for studies of a variety of cell biological processes, the identification and cloning of genes known by mutations is still arduous. Current methods are inefficient for many of the existing mutations as their mutant alleles can either spontaneously revert or produce no selectable phenotype. With the sequencing and the annotation of the C. reinhardtii nuclear genome and mapping of available molecular markers, positional cloning is now possible. This thesis explored the application of bulked segregant analysis (BSA) with two types of molecular markers, amplified fragment length polymorphisms and single nucleotide polymorphisms, in order to identify and characterize the nuclear TBC1 gene and its predicted product. TBC1 was previously shown to functionally interact with specific regions and structures in the 5' untranslated region of chloroplast psbC mRNA to promote its translation and the synthesis of its product, the CP43 subunit polypeptide of PSII. Using BSA, TBC1 was mapped to an 8 map-unit region of Linkage Group VI. Complementation analysis narrowed down its position to a 41 kb region. Analyses of predicted genes in the region identified an exonuclease II orthologue as the best candidate for being TBC1