Dissertations / Theses on the topic 'Transcript variants'

Genome Wide Association Studies (GWAS) has identified HNF1B located at chromosome 17q12 as foremost risk gene for prostate cancer susceptibility in multi-ethnic populations. This thesis characterises the HNF1B transcript variants along with defining their expression pattern and functional attributes in prostate cancer. Future research will enable the discovery of the molecular mechanism of their actions and whether targeting these HNF1B transcript variants in cancer may prove a useful therapeutic strategy.

BRAF is a widely studied oncogene and its functions are well characterized in several cellular contests and diseases. However the regulation of the expression of BRAF is mostly unknown. With the aim to understand the post-transcriptional regulation of BRAF, we performed 3’RACE in A375 melanoma cells and we found 2 different 3’UTRs: the one commonly reported in many data bases (Reference) and a new one that is only predicted (X1). The two 3’UTRs are completely different in sequence and length (120nt vs 1350nt). Furthermore, they are transcribed from exon 18 or thanks to an alternative splicing event occurring between exon 18 and a newly discovered exon 19 (X1). By using Real Time PCR, we confirmed the expression of both transcripts in melanoma and non-melanoma cell lines. Moreover, using RNA-SEQ data available at TCGA, we showed the co-expression of Reference and X1 BRAF transcripts also in human biopsies. Due to our discovery that BRAF exists in at least 2 different transcript variants, we decided to investigate further the expression of all the BRAF isoforms reported in NCBI and Ensembl. To do so, we took advantage of the RNA-seq data of more than 4,800 patients belonging to 9 different cancer types. We show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1 and BRAF-X2) that differ in the last part of their open reading frames, as well as in the length (BRAF-ref: 76nt; BRAF-X1 and X2: up to 7kb) and in the sequence of their 3’UTRs. In melanoma cells, the X1 isoform is expressed at the highest level, while the most prevalent among the three isoforms varies from one cancer type to another. Moreover, the relative abundance among the three BRAF isoforms is maintained in melanoma cells with acquired resistance to BRAF and MEK inhibitors driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. Besides their 3’UTRs, also the very last part of the coding sequences differ among the three isoforms. By immunoprecipitation of BRAF in A375 cells and subsequently Mass-SPEC analysis, we revealed the existence of Reference and X1 proteins which are expressed at similar levels, while X2 is not detectable because quicky degraded by the proteasome. Furthermore functional studies show that the two proteins account together for BRAF activities both in vitro and in vivo. Given the differences in length and sequence between the reference and the X1 3’UTR, we hypothesized that the two isoforms undergo different regulation mediated by RNA-binding proteins or non-coding RNAs. We focused on post-transcriptional regulation by microRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the expression of target messenger RNAs (mRNAs) and for this reason play a key role in virtually all cellular processes. In spite of the availability of several prediction algorithms, the identification of specific miRNA-target interactions remains a challenge. In order to overcome this problem we developed an innovative method, called miR-CATCH v2.0, for the high-throughput identification of microRNAs that bind a target transcript. The protocol is based on the affinity purification of the target mRNA and bound miRNAs by using two different pools of 3’biotinylated anti-sense DNA probes (ODD and EVEN). We designed 12 probes (6 ODD probes and 6 EVEN probes) for the purification of X1-3’UTR-miRNAs complexes and we performed three separate and independent captures in A375 metastatic melanoma cells. MicroRNAs were identified through small RNA-sequencing and the top-scoring miRNAs that resulted consistently enriched in all the captures will be validated in vitro and in vivo experiments.

Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an in vitro model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination.

Two cell clones were chosen, a GFAP⁺ and a GFAP^-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified.

PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene v-sis. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to v-sis thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.

Teneurins are transmembrane glycoproteins encoded by ODZ genes. Teneurins are a unique protein family conserved from flies and worms to human and are mainly expressed in the developing and adult nervous system where they are thought to be crucial for neurogenesis and axon-guidance. Teneurins are also expressed outside CNS where they have been proposed to play a role in morphogenesis and cell migration. Vertebrate teneurin expression pattern has been studied most extensively in mice and chicken, however still very little is known about their biological function and mechanism of action in humans. Moreover, experimental data concerning the molecular structure of vertebrate teneurins transcripts is scarce, and human ODZ4 messenger has not been subject to a detailed characterization before. Furthermore, recent studies have evidenced that some ODZs, specifically ODZ2 and 4, could be involved in tumor development in a still unclear mode. To this respect, in our preliminary study we demonstrated the expression of ODZ2 in human ovarian and breast cancer-derived cell lines by RT-PCR. Therefore, in this thesis work we have evaluated the expression of ODZ4 in human ovarian and breast cancer-derived cell lines by RT-PCR. As a result, we have characterized two partial ODZ4 full-length messengers expressed by these cell types. The exon-expression pattern analysis indicates that these ODZ4 transcripts can be differentiated by the presence of an insert region corresponding to a non-adjacent genomic sequence that lies between exons 6 and 7. Additionally, the number of exons expressed differs depending on the type of cell line analyzed, even though they may derive from the same tissue type. Further, exonic deletions were not detected along these ODZ4 transcripts but many other truncated splice variants were observed. This study intends to generate new molecular information necessary for the elucidation of the functional role of ODZ4 in human cancer.

In the past decade, new concepts have emerged on the role of adhesion molecules in the communication, motility, differentiation, and survival of cells in the body. The traditional view on the role of cell adhesion molecules in cell differentiation and tissue architecture has changed following the discovery that some adhesion proteins, including CD44, are involved in tumor progression in both animal and human tissues. CD44 is a glycoprotein which has differently spliced isoforms with various combinations of up to 9 variant exons numbered v2 to v10. The aim of this study was to characterize CD44 transcripts associated with colorectal neoplasia. The hypothesis is that discrete transcripts of CD44 are generated during the progression from normal to carcinomatous colonic epithelium, contributing to determinant changes in cell adhesion. Our results showed: (i) a high level of expression of CD44 v8-v10 transcripts in all tumor cells by in situ hybridization technique, (ii) 4 transcripts of CD44v7 with 650, 740, 1000, and 1150 bases in RNA extracted from tumor cases and studied by RT-PCR followed by Southern blotting, and (iii) the 1150 transcript was the only one found in both carcinoma and polyps. This transcript has therefore the potential to be used as an early marker of progression in colorectal neoplasia. It is reasonable to suggest that these new transcript species generate protein isoforms with different adhesion properties, as compared to standard CD44.

La pigmentation chez les mammifères est définie par la distribution ainsi que par les quantités relatives de deux pigments, la phaléomélanine et l'eumélanine qui produisent respectivement des colorations rouge/jaune et marron/noir. Aujourd'hui, plus de 150 mutations différentes affectant plus de 60 loci distincts modifiant la coloration du pelage ont été identifiées chez la souris. Deux principaux loci, agouti (a) et extension (E) contrôlent les quantités relatives des pigments. Le gène extension code le récepteur (MClr) de l'hormone hypophysaire α-MSH. Cette dernière stimule la production d'eumélanine en se fixant sur le récepteur ancré dans la menbrane des mélanocytes. Par sa liaison à MClr, la protéine Agouti favorise la synthèse de la phaléomélanine. Le travail présenté dans ce mémoire a été entrepris dans le cadre d'un programme de recherche de marqueurs génétiques des races bovines françaises en collaboration avec les centres de recherche de l'INRA et les UPRA. L'étude des exons codants du gène agouti bovin n'a pas permis de mettre en évidence l'existence de différents allèles associés aux races bovines étudiées. Aussi, avons nous orientés nos travaux de recherche vers lé́tude des transcrits du gène agouti afin d'expliquer les variations de pigmentation de la robe des races bovines étudiées. Ces recherches nous ont conduit à identifier, en plus du transcrit bovin de référence, deux transcrits spécifiques de la peau. Ces transcrits diffèrent dans leurs structures et leur séquence, et définissent deux groupes de races bovines: le premier comprend les races Prim'Holstein noire, Limousine X prim'Holstein, Montbéliarde et Limousine et le deuxième les races Prim'holstein blanche, charolaise X Prim'Holstein, Blonde d'Aquitaine, Charolaise et Salers. La prodution par génie génétique des pépdides deduits des transcrits bovins permettra d'obtenir des anticorps spécifiques. Ces derniers seront utilisés dans des études d'immunodetection. La mise en évidence de sites d'épissage alternatifs au sein des transcrits spécifiques de la peau et l'obtention des séquences 5' non-codantes ont également conduit à établir la première structure partielle du gène Agouti bovin
The work presented in this report was begun within the framework of a research program for genetic markers of the French bovine species in association with INRA and UPRA. .

RNA transcripts are expressed from tens of thousands of loci across the human genome. Several studies have suggested that many genes are alternatively expressed to produced multiple mRNA isoforms and many of these remain undiscovered. Identifying specific isoforms associated with human diseases such as cancer has potential to lead to improved treatments. The scale and complexity of the transcriptome present significant barriers to (1) identifying isoforms and (2) applying knowledge to human disease research. Recent advances in genome-wide microarray and sequencing platforms have begun to provide the capacity and resolution to address these challenges. The goal of this thesis was to develop novel methods that allow genome-wide identification and quantification of mRNA isoforms. I first approached this problem by creating a microarray design platform for alternative expression analysis called 'ALEXA-array' (www.AlexaPlatform.org). To evaluate the ALEXA-array approach I used it to generate a microarray design that I then used to measure differential expression of mRNA isoforms in 5-fluorouracil (5-FU) sensitive and resistant colorectal cancer cell lines. This approach identified several isoforms potentially involved in 5-FU resistance. While the ALEXA-array approach was successful, I identified several limitations of the method. For example, the approach was insensitive to isoforms with small differences in sequence content and limited by both the transcriptome annotations and the number of microarray features available at design time. I developed a second method, ‘ALEXA-seq’, to take advantage of advances in massively parallel sequencing. Applying this method to the same cell lines I showed that the approach was able to overcome many limitations of the microarray approach. Several additional candidate 5-FU resistance isoforms were identified. Both the ALEXA-array and ALEXA-seq approaches identified expression of an aberrant isoform of the uridine monophosphate synthetase as a top candidate. Interestingly, this gene was suspected to function in the conversion of 5-FU to active anti-cancer metabolites. Additional characterization was performed to elucidate the expression pattern, transcript diversity and sequence variation of this gene in a panel of cell lines and tumours. The methods presented here should help to identify mRNA isoforms with potential utility as therapeutic targets or as prognostic or diagnostic markers.

Alternative splicing of the zebrafish Myosin Phosphatase Targeting Subunit, MYPT1, produces a novel isoform (TV202). TV202 and the truncated TV202Δ ere shown to form an active complex with Protein Phosphatase 1 β (PP1β) via stress fiber assay. TV202 was also shown to be localized in the cytoplasm, enriched in a paranuclear manner. TV202Δ was found the be localized inside the nucleus. It was also found that TV202 was zygotically, but not maternally, expressed during early zebrafish development via RT-PCR.

En 2017, l'Assemblée mondiale de la Santé et l'Organisation mondiale de la Santé ont fait du sepsis une priorité de santé mondiale en adoptant une résolution pour améliorer, prévenir, diagnostiquer et gérer le sepsis. Cette pathologie tue principalement des personnes déjà fragilisées. Il a été démontré que l'état du patient et la réponse immunitaire de l'hôte est primordiale dans le développement de la pathologie. Mon projet de thèse a tout d’abord consisté à caractériser la réponse transcriptionnelle de cellules du péritoine stimulées au LPS dans un modèle murin du sepsis. Par ailleurs, j’ai recherché les variants génétiques cis-régulateurs potentiellement impliqués dans la mortalité des patients en choc septique par une approche bioinformatique et une validation fonctionnelle expérimentale. Ces 2 projets mettent en évidence, chez l'homme, des mécanismes de la régulation de l’expression de gènes clés dans la survie des patients atteints de sepsis
In 2017, the World Health Assembly and the World Health Organization made sepsis a global health priority by adopting a resolution to improve, prevent, diagnose and manage sepsis. This pathology mainly kills people who are already vulnerable. It has been shown that the patient's condition and the host's immune response are essential in the development of the disease. My thesis project first consisted in characterizing the transcriptional response of peritoneal cells stimulated at the LPS in a mouse model of sepsis. In addition, I researched cis-regulatory genetic variants potentially involved in the mortality of patients in septic shock through a bioinformatics approach and experimental functional validation. These 2 projects highlight, in humans, mechanisms for regulating the expression of key genes in the survival of patients with sepsis

Les gènes du chromosomeX sont majoritairement inactivés au cours de la méiose mâle. Chez la souris, seulement 6% d’entre eux sont réactivés au cours des stades post-méiotiques. Parmi eux le gène Uba1X codant pour l’enzyme activatrice de l'ubiquitine, UBA1 qui produit trois transcrits dont deux sont ubiquitaires mais le troisième prédomine dans les cellules post¬-méiotiques : les spermatides. Nos travaux montrent que les 5’UTR, seul différence entre ces trois transcrits, déterminent la localisation et la dose relative des isoformes nucléaire et cytoplasmique de la protéine UBA1. Nous avons mis en évidence chez la souris que le transcrit spermatide-spécifique code pour l'isoforme nucléaire, exprimée fortement dans les spermatides suggérant un rôle de la protéine UBA1 dans la dégradation des histones lors du remodelage chromatinien. Nous avons détecté deux mutations dans la région spermatide-spécifique du gène : une délétion de 13pb et une transition G>A, chacune portée par un patient infertile, et non retrouvée dans notre population témoin. Les analyses ont montré que la délétion de 13pb induit un épissage anormal du transcrit spermatide-spécifique et que la transition G>A pouvait réduire le taux d’expression du transcrit spermatide-spécifique. Ces mutations pourraient induire l'infertilité des deux patients. En parallèle nous avons pu démontrer que les mutations dans le gène codant pour la protamine PRM1 décrites dans la littérature c.102G>T et c.-107G>C ne sont pas liées à l'infertilité masculine et que le variant est un polymorphisme fréquemment retrouvé dans la population congolaise
The majority of genes on the X chromosome are repressed during meiosis and only 6% of them are expressed in post meiotic germ cells. One of these genes is Ubal, encoding the ubiquitin-activating enzyme UBA1. Ubal produces three different transcripts, two of which are ubiquitously expressed while the third is predominant in the post meiotic germ cells: the spermatids. Our study shows that the 5’UTR, which is the only difference between these transcripts, determines the localization and the relative dose of the nuclear and cytoplasmic isoform of the UBA1 protein. The spermatid-specific transcript encodes for the nuclear isoform in the spermatids in the mouse suggesting that the UBA1 protein is implicated in chromatin remodeling during spermiogenesis. We have detected two mutations in the spermatid-specific region of the UBA1 gene in two infertile men: a deletion of 13bp and a G>A transition, neither of which was found in our cohort of fertile men. The deletion of 13bp diminishes the correct splicing of the spermatid-specific transcript and that the G>A transition may reduce expression of the spermatid-specific transcript. These results show that the UBA1 gene is involved in spermiogenesis, and reactivated in spermatids by its spermatid-specific transcript and that the mutations identified may induce infertility by reducing UBA1 levels in spermatids. We have also demonstrated that two variants described in the protamine codant gene PRM1C.102G>T and c.-107G>C are clearly not associated with male infertility and that the c.-107G>C is polymorphism frequently found in the congolese population

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

端粒酶是很重要去保護端粒的長度，它會表達於包括癌細胞與幹細胞在內的高度增殖細胞，用以解答這些細胞的無限分化能力。端粒酶包括兩個組成部分:由核糖核酸組成的部分和名為逆轉錄酶(TERT)組成的部分。近年，Pin2/TRF1結合蛋白(PinX1)被發現能透過在C末端的端粒酶抑制區域和逆轉錄酶作直接的交互作用以達到它作為腫瘤抑制蛋白的目的。但是，PinX1在幹細胞上的作用並沒有被探討過。
我的研究最近發現老鼠PinX1(mPinX1)能在老鼠幹細胞(mESCs)上表達。另外我亦在mESCs上發現了一種相比mPinX1缺乏了C末端名為mPinX1t的全新轉錄變異。有趣地，在幹細胞的分化過程中，mPinX1的表達隨着分化過程下跌而mPinX1t的表達隨着分化過程上升。mPinX1和mPinX1t的表達變化並沒有影響幹細胞在未分化時的狀態﹔但在細胞分化的過程中，透過心肌特定基因的表達反映中出當降低及增加mPinX1的表達會抑制心肌細胞分化，而增加mPinX1t的表達會增長心肌細胞分化。我的結果清楚反映出mPinX1和mPinX1t的表達決定了mESCs的心肌細胞分化。去驗證mPinX1t在蛋白上表達，我利用了核糖體分離檢定和 5’-RACE檢定，結果顯示mPinX1t的mRNA是積極地被轉錄成為蛋白質。
為了找出mPinX1和mPinX1t影響心肌細胞分化的分子機制，我透過RNA免疫沉澱法和即時定量聚合酶鏈鎖反應去檢測mPinX1和mPinX1t會否和心肌轉錄因子（Gata4/Tbx5）的mRNA有交互作用。結果顯示mPinX1和mPinX1t皆能和研究中的心肌轉錄因子的mRNA有交互作用，代表mPinX1和mPinX1t可能透過此交互作用去影響心肌細胞分化。另外，透過酵母菌雙雜合系統檢定，我找出一些和mPinX1t有可能結合的蛋白。這些和mPinX1t有可能結合的蛋白根於基因本體論被分類成不同種類。我亦透過蛋白質免疫沉澱法去驗証這些交互作用。而其中一個和mPinX1t結合的蛋白名為Parva, 它會和PINCH及Integrin linked kinase (ILK)組成複合物從而影響肌小節的形成。我在免疫細胞化學實驗中和對照實驗比較發現在敲除mPinX1/mPinX1t基因的mESCs，心肌分化過程中肌小節形成有延遲。這說明了mPinX1t對肌小節形成有正面作用。
我發現了mPinX1和mPinX1t在幹細胞上對於心肌細胞分化的新作用。這次研究除了讓我們了解幹細胞的基礎生物學，同時亦給予我們重要的遠見去解決關於幹細胞分化心肌細胞在醫療上的困難：怎樣去增加心肌細胞的數量。找出和mPinX1t有交互作用的蛋白的發現可以讓我們找到mPinX1t影響心肌細胞分化的秘密。
Telomerase is important to maintain the length of telomere. It is expressed in highly proliferating cells such as cancer cells and embryonic stem cells (ESCs), accounting for their unlimited proliferation capacity. Telomerase consists of two components: RNA and reverse transcriptase component, known as telomerase reverse transcriptase (TERT). Recently, Pin2/TRF1 interacting protein (PinX1) has been found to interact directly with TERT through its telomerase inhibitory domain at its C terminal and act as a putative tumor suppressor. However, the role of PinX1 in ESCs has never been explored.
In my current study, I have found that the mouse PinX1 (mPinX1) protein is expressed in mouse ESCs (mESCs). Additionally, a novel transcript variant mouse PinX1t (mPinX1t), which is predicted to encode a protein lacking the C terminal of mPinX1, was detected in mESCs. Interestingly, expression of mPinX1 decreased while that of mPinX1t increased during mESC differentiation. Altering the expression of mPinX1 and mPinX1t did not affect mESCs at undifferentiated state. Importantly, upon differentiation, altering (both knocking down or overexpressing) the expression of mPinX1 decreased the cardiac differentiation while overexpressing mPinX1t increased the cardiac differentiation as revealed by the expression of cardiac-specific genes. The results clearly indicate that expression of mPinX1 and mPinX1t determines the cardiac fate of mESCs. To validate the protein expression of mPinX1t, polysome fractionation and 5’-RACE assay have been performed and the results showed that mPinX1t was an actively translating mRNA.
To find out the molecular mechanism of how mPinX1 and mPinX1t regulate cardiac differentiation, RNA immunoprecipitation assay and qPCR have been performed to look at the interactions between mPinX1/mPinX1t and the mRNAs of cardiac transcription factors (Gata4/Tbx5). The results showed that both mPinX1 and mPinX1t bind to the mRNAs of all the cardiac transcription factors under investigation, suggesting that mPinX1/mPinX1t proteins may affect cardiac differentiation through these interactions. In addition, possible protein interacting partners of mPinX1t have been determined using yeast two hybrid assay. These interacting partners were classified based on gene ontology (GO) terms. Further co-immunoprecipitation assay was carried out to validate the interactions. One of the interacting partners was Parva, which formed a complex with PINCH and Integrin linked kinase (ILK) to regulate sarcomere assembly. In mPinX1/mPinX1t knockdown, there was a delay in sarcomere formation in early differentiation when compared to the scrambled control by immunocytochemistry assay, suggesting the positive role of mPinX1t on sarcomere assembly.
My study reports the novel role of mPinX1 and mPinX1t in determining the cardiac differentiation potential of mESCs. This study will provide interesting information not only for the basic biology of mESCs, it may also provide important insights for overcoming one of the hurdles of using mESC-derived cardiomyocytes in future therapy: how to increase the yield of cardiomyocytes. The discoveries of mPinX1t interacting partners help provide insight on the molecular mysteries on how mPinX1t regulates cardiac differentiation.
Chan, Hing Chung.
Thesis Ph.D. Chinese University of Hong Kong 2016.
Includes bibliographical references (leaves ).
Abstracts also in Chinese.
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Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus.
PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014