C-reactive protein (CRP) is synthesized in hepatocytes. The serum concentration of CRP increases dramatically during the acute phase response. In human hepatoma Hep3B cells, maximal CRP expression occurs in cells treated with the combination of IL-6 and IL-1β. IL-6 induces transcription of the CRP gene and IL-1β synergistically enhances the effects of IL-6. We investigated the role of IL-6-activated transcription factor STAT3, also known as STAT3α, in inducing CRP expression since we identified four consensus STAT3-binding sites centered at positions - 72, - 108, - 134 and - 164 on the CRP promoter. It has been shown previously that STAT3 binds to the site at - 108 and induces CRP expression. We found that STAT3 also bound to the other three sites, and several STAT3-containing complexes were formed at each site, suggesting the presence of STAT3 isoforms and additional transcription factors in the complexes. Mutation of the STAT3 sites at - 108, - 134 or - 164 resulted in decreased CRP expression in response to IL-6 and IL-1β treatment, although the synergy between IL-6 and IL-1β was not affected by the mutations. The STAT3 site at - 72 could not be investigated employing mutagenesis. We also found that IL-6 activated two isoforms of STAT3 in Hep3B cells: STAT3α which contains both a DNA-binding domain and a transactivation domain and STAT3β which contains only the DNA-binding domain. Taken together, these findings raise the possibility that IL-6 not only induces CRP expression but also regulates the induction of CRP expression by activating STAT3 isoforms and by utilizing all four STAT3 sites.

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of iv T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7- deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bimcontaining T-cells, indicating that in these cells autophagy was protective. IL-7 dependent Tcells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosometracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy

The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Forster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

No / Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

Tumor suppressor p53, the guardian of the genome, has been intensely studied molecule owing to its central role in maintaining cellular integrity. While the level of p53 protein is maintained low in unstressed conditions, there is a rapid increase in the functional p53 protein levels during stress conditions. It is now well documented in literature that p53 protein accumulates in the cells following DNA damage by posttranslational modifications leading to increased stability and half life of protein. Additionally, recent studies have also highlighted the significance of increased p53 translation during stress conditions. Interestingly, an alternative initiation codon has been shown to be present within the coding region of p53 mRNA. Translation initiation from this internal AUG results in an N-terminally truncated p53 isoform, described as ΔN-p53. However, the mechanisms underlying co-translational regulation of p53 and ΔN-p53 are still poorly understood. Studies have suggested that synthesis of both p53 and its ΔN-p53 isoform is regulated during cell cycle and also stress and cell-type specific manner. Interestingly, reports also demonstrate continued synthesis of both p53 isoforms during stress conditions. In contrast, global rates of cap-dependent translation initiation are shown to be reduced during stress conditions. This translation attenuation is observed mainly due to restricted availability of critical initiation factors. Interestingly, preferential synthesis of a vital pool of survival factors persists even during these circumstances. Studies have suggested that this selective translation is mediated via alternative mechanisms of translation initiation. One of the important mechanisms used for protein synthesis during these conditions is internal initiation. In this mechanism, the ribosomes are recruited to a complex RNA structural element known as ‘Internal Ribosome Entry Site (IRES)’, generally present in the 5’ untranslated region (UTR) of mRNA. Therefore, it is possible that the translation of p53 and ΔN-p53 could also be regulated by IRES mediated translation, especially during stress conditions. In this thesis the role of internal initiation in translational control of p53 and ΔN-p53 has been investigated. Additionally, the putative secondary structure of p53 IRES RNA has been determined. Further, it has been shown that polypyrimidine tract binding (PTB) protein acts as an important regulator of p53 IRES activities. The probable mechanism of action of PTB protein has also been investigated. The results suggest that interaction with PTB alters the p53 IRES conformation which could facilitate translation initiation. Finally, the possible physiological significance of existence of p53 IRES elements has been addressed. In the first part of the thesis, the presence of internal ribosome entry site within p53 mRNA has been investigated. As a first step, the 5’UTRs mediating the translation of both p53 and ΔN-p53 were cloned in the intercistronic regions of bicistronic constructs. Results of in vivo transfection of these bicistronic constructs suggested the presence of two IRES elements within p53 mRNA, with activities comparable to known viral and cellular IRESs. The IRES directing the translation of p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of ΔN-p53 extends further into the protein-coding region. To further validate, stringent assays were performed to rule out the possibility of any cryptic promoter activity, re-initiation/scanning or alternative splicing in the p53 mRNA. Transfection of in vitro synthesized bicistronic RNAs confirmed the presence of IRES elements within p53 mRNA. Incidentally, this constitutes the first report on translational control of p53 by internal initiation. In the second part of the thesis, the secondary structure of p53 IRES RNA has been investigated. Structural analysis of p53 RNA was performed using structure-specific nucleases and modifying chemicals. The results obtained from chemical modification and nuclease probing experiments were used to constrain Mfold predicted structures. Based on this, a putative secondary structure model for p53 IRES RNA has been derived. Sequence alignment suggested that the p53 IRES RNA showed significant sequence conservation across mammalian species. To study the effect of mutations on the IRES structure, mutant p53 IRESs were used that harbor silent mutations at critical locations within the p53 IRES element. Incidentally, one of the mutant constructs used in the study was observed to be a naturally occurring mutation in a chronic lymphocyte leukemia patient. RNA structure analyses of these two mutant p53 IRES RNAs were performed. The nuclease mapping data suggested conformational alteration in these mutant RNAs with respect to wild type. Consistently, a comparative Circular-Dichroism spectroscopy of the Wt and mutant RNAs also validated the conformational alteration of the mutant RNAs. This also suggested that the presence of mutations in p53 IRES might result in decreased induction of p53 protein following DNA damage due to altered RNA structure. This might constitute as one of the mechanisms leading to tumor development in some types of cancers. In the third part of the thesis, the role of important cellular proteins that might modulate p53 IRES mediated translation has been studied. These cellular proteins act as IRES interacting trans-acting factors (ITAFs). Polypyrimidine tract binding (PTB) protein is an important ITAF implicated in regulating IRES mediated gene expression during apoptosis. It was observed that PTB protein specifically interacts with both the IRES elements within p53 mRNA. Interestingly, the affinity of interaction of PTB protein with both p53 IRES RNAs was observed to be significantly different. In order to determine the contact points of PTB on p53 IRES, a foot-printing assay using structure specific nuclease and recombinant-PTB protein was performed on p53 RNA. The data from foot-printing as well as primer extension inhibition assay (toe-printing analysis) suggested the presence of multiple PTB binding sites on p53 IRES RNA. Based on these results, a deletion mutant was generated that showed reduced PTB binding and also reduced IRES activity as compared to wild type. Further, to study the role of PTB in mediating p53 translation, the expression of PTB gene was partially silenced by using PTB specific siRNA. Partial depletion of endogenous PTB protein showed a significant decrease in the p53 IRES activities. These results suggest that PTB protein is essential for the p53 IRES activities. To understand the probable mechanism by which PTB regulates p53 IRES mediated translation, CD spectroscopy analysis of p53 IRES RNA was performed in the absence and presence of PTB protein. Interestingly, CD spectra analysis of the p53 RNA in the presence of PTB suggested a specific conformational change in p53 IRES, which might probably facilitate ribosome loading during internal initiation. This also suggests that abnormal expression of p53 ITAFs might lead to reduced p53 induction following DNA damage conditions. It could also be another event leading to malignant transformation of cells bearing wild type p53. It is highly tempting to speculate that the levels of p53 ITAFs could also be used as tumor biomarkers. In the fourth part of the thesis, the physiological relevance of existence of IRES elements within p53 mRNA has been investigated. The levels of p53 and ΔN-p53 proteins are known to be regulated in a cell cycle phase-dependent manner. The IRES activities of both p53 IRES elements were investigated at different phases of cell cycle. The activity of the IRES responsible for translation of p53 protein was found to be highest at G2-M transition and the maximum IRES activity corresponding to ΔN-p53 synthesis was observed at G1-S transition. These results suggested that the p53 IRES activities are regulated in a cell-cycle phase-dependent manner. Next, the regulation of p53 IRES mediated translation during stress conditions was studied. Human lung carcinoma cell line, A549 cells (that endogenously express both the p53 isoforms), were exposed to DNA damaging drug, doxorubicin. The level of p53 protein was observed to increase in a time-dependent manner. Interestingly, PTB protein, which is predominantly nuclear, was found to translocate to the cytoplasm during stress condition in a time-dependent manner. Under similar conditions, p53 protein was observed to reverse translocate from the cytoplasm to nucleus, probably to function as a transcription factor. Next, the influence of partial PTB silencing on p53 isoforms in the presence of cell stress (mediated by doxorubicin) was investigated. The data indicated reduced levels of both p53 and ΔN-p53 when PTB gene expression was partially silenced. These observations constitute “the proof of concept” that relative abundance of an ITAF, such as PTB protein, might contribute to regulating the coordinated expression of the p53 isoforms. The thesis reveals the presence as well as the physiological relevance of existence of IRES elements within p53 mRNA. The novel discovery of p53 IRES elements may provide new insights into the underlying mechanism of translational regulation. The modulation of the p53 IRES activities by PTB protein suggests that the regulated expression of p53 isoforms depends on the integrity of IRES elements and availability of cellular proteins that can serve as p53 ITAFs. Thus, studies pertaining to the identification of mutations within p53 IRES region as well as abnormal expression of p53 ITAFs such as PTB in cancer cells may have far reaching implications. These studies might lead to further advances in the field of cancer detection, prognosis and design of novel therapeutic strategies.

Abstract Collagen XVIII is an evolutionarily conserved, ubiquitously-expressed basement membrane (BM) proteoglycan produced in three isoforms, the individual roles of which are largely unknown. The physiological in vivo roles of these collagen XVIII isoforms are studied here using novel genetically modified mouse strains deficient in either the short or the medium/long isoforms of the molecule. In addition, the effects of keratin-14-driven overexpression of the thrombospondin-1 (Tsp-1) –like domain, which is common to all three collagen XVIII isoforms, are studied. The findings underline the importance of the short collagen XVIII isoform in the eye, as its absence was sufficient to cause the aberrant vascularisation of the retina previously reported in mice lacking all isoforms of collagen XVIII. In addition, an excess of the collagen XVIII Tsp-1 domain led to serious eye abnormalities, possibly by interfering with the functions of the full-length collagen XVIII produced in mice. Collagen XVIII was also shown to contribute to adipogenesis in an isoform-specific manner, in that a lack of the medium/long isoforms of collagen XVIII led to impaired adipocyte maturation and the subsequent reduction in the adipocyte number induced liver steatosis and hypertriglyceridaemia. Hence this work establishes a new extracellular matrix ECM-directed mechanism contributing to control over the multistep adipogenesis programme and points to the functional consequences of its impairment for ectopic fat deposition. The enzymatic remodelling of ECM components results in molecules with novel biological activities. Arresten is a collagen IV(α1)-derived fragment with anti-angiogenic properties which was originally described as not having any direct anti-tumour effects on cancer cells themselves. The present data revealed novel inhibitory roles for arresten in oral squamous carcinoma cell proliferation, survival, motility and invasion. Since arresten is a potent inhibitor of angiogenesis, the data generated here further underline the possibility for using it as a therapeutic agent in cases of cancer. / Tiivistelmä Kollageeni XVIII on tyvikalvojen proteoglykaani ja yksi harvoista evoluutiossa konservoituneista kollageeneista. Se esiintyy elimistössä kolmena isomuotona, joiden biologiset tehtävät ovat vielä jokseenkin epäselviä. Tässä tutkimuksessa selvitettiin kollageeni XVIII:n isomuotojen fysiologista merkitystä hyödyntäen uusia hiirilinjoja, joilta kollageeni XVIII:n lyhyt tai kaksi pisintä varianttia oli geneettisesti inaktivoitu. Poistogeenisten hiirimallien rinnalle tehtiin kaikille varianteille yhteistä trombospondiini-1 (Tsp-1)-domeinia yli-ilmentävä hiirilinja. Tämän väitöskirjatutkimuksen avulla saatiin uutta tietoa kollageeni XVIII:n ja etenkin sen lyhimmän variantin tärkeästä roolista silmässä. Aikaisemmat tutkimukset ovat osoittaneet kollageeni XVIII:n puutteen häiritsevän silmän verkkokalvon verisuonituksen normaalia kehittymistä. Tässä työssä havaittiin, että pelkästään lyhyen isomuodon puute riitti altistamaan hiiret muutoksille verkkokalvon suonituksessa. Tsp-1-osan ylimäärän havaittiin lisäksi alistavan hiiret muutoksille silmän rakenteessa, mahdollisesti häiritsemällä silmässä jo olemassa olevan kollageeni XVIII:n toimintaa. Tässä työssä havaittiin myös uusi yhteys kollageeni XVIII:n ja rasvasolujen kypsymisen välillä. Verrokkihiiriin verrattuna muodostuvan rasvakudoksen havaittiin jäävän merkittävästi vähäisemmäksi poistogeenisillä hiirillä, joilta kollageeni XVIII:n pitkät isomuodot olivat geneettisesti inaktivoitu. Heikentynyt rasvakudoksen muodostuminen lisäsi triglyseridien kertymistä hiiren verenkiertoon ja maksaan. Tutkimustulos on merkittävä avaus soluväliaineen merkityksestä rasva-aineenvaihdunnalle ja kannustaa lisätutkimuksilla selvittämään, onko kollageeni XVIII:lla yhteys myös ihmisen metaboliseen oireyhtymään. Soluväliaineen komponenttien entsymaattinen muokkaus tuottaa usein molekyylejä, joilla on uusia isäntämolekyyleistä poikkeavia ominaisuuksia. Tässä työssä tutkittiin yhden tällaisen molekyylin, tyvikalvokollageenin IV hajoamistuotteen, arrestenin, suoria vaikutuksia syöpäsoluille. Arrestenin tiedettiin entuudestaan estävän syöpäkasvainten verisuonten uudismuodostusta koe-eläimillä. Työssä osoitettiin, että arresten vaikutti endoteelisolujen lisäksi myös itse syöpäsoluihin estäen niiden lisääntymistä ja vähentäen niiden elinkykyä ja liikkuvuutta, mikä tekee arrestenista entistä houkuttelevamman ehdokasmolekyylin lääkekehitystyöhön.

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.

No / Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5 mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real-time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2',7' Dichlorodihydrofluorescein Diacetate (DCFH-DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC versus c.HUVEC (>1.5-fold change; P < 0.05). Functional clustering analysis of these differentially expressed genes revealed participation in inflammatory responses (including adhesion) which may be related to pathological outcomes. Of these genes, ICAM-1 was validated as upregulated, confirming microarray results. Additional confirmatory immunofluorescence staining revealed increased protein expression of ICAM-1 compared with c.HUVEC which was reduced by vitamin C treatment (100 muM). Thus, maternal diabetes induces persistent alterations in fetal endothelial function and gene expression following glucose normalization and antioxidant treatment could help reverse endothelium dysfunction.

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de > 440 kDa e ~230 kDa, e de ~140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. Análises por imunofluorescência e microscopia imunoeletrônica sugerem que poliductina se localize em cílio apical primário, membrana apical e citoplasma de células do ducto coletor. Nossos resultados indicam que PKHD1 codifica isoformas de membrana e solúveis / PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of > 440 kDa and 230 kDa, and of ~140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland. Immunofluorescence and immunoelectron microscopy studies suggest that polyductin localizes to primary apical cilium, apical membrane and cytoplasm of collecting duct cells. Our data indicate that PKHD1 codifies membrane and soluble isoforms