Adipose tissues have long been considered to derive from a common origin. Even the functionally different brown and white adipose tissues were generalized to share a common origin. Brown adipose tissue is a highly innervated and vascularised tissue containing multilocular and multimitochondrial brown adipocytes. Brown adipose tissue expends energy through sympathetic nervous system-mediated non-shivering thermogenesis, where uncoupling protein 1 (UCP1) is the key player. In contrast, white adipose tissue consists of unilocular white adipocytes with a main role to store energy in the form of the lipid droplet. We know today that this generalisation is exaggerated since adipocytes can derive from more than one origin and not only be brown or white. We and others have demonstrated that the brown adipocyte has a dermomyotomal origin and derives from the adipomyocyte, the precursor cell that can also become a myocyte, whereas white adipocytes are suggested to derive from pericytes, cells that are embedded within the vascular vessel walls. For a long time there has been evidence that energy-expending adipocytes reside within certain white adipose tissues, based on the fact that cold exposure, by switching on the sympathetic nervous system, leads to levels of UCP1 that are not detectable in mice housed at thermoneutrality. We demonstrated that these cells have a molecular signature that is distinct from brown and white adipocytes. Since these energy-expending cells reside within certain white adipose tissues, we chose to name them brite (brown in white) adipocytes. Moreover, we also identified regulatory factors that were specifically expressed in each adipocyte type, thus, facilitating the possibility to identify the three adipocytes: the brown, the white and the brite. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

<p>In recent years, the pervasiveness and importance of post-transcriptional regulation has reshaped the underlying principles of the organizational logic of gene expression. RNA-binding proteins (RBPs) and non-coding RNAs are the regulatory molecules primary responsible for interaction with target mRNAs and thereby regulating post-transcriptional processes eventually influencing characteristics of the encoded protein. Many of the mRNA targets of RBPs encode functionally related proteins, which for post-trascriptional operons, resulting in coordination of macromolecular complexes or specific cellular processes. Thus, identifying RNA targets, precise binding sites, and the dynamics of these interactions will reveal how these important regulatory factors contribute to gene regulatory networks.</p><p>ELAV family of human RBPs consist of 4 members, which all have 3 RRM (RNA-recognition motif) domains the last separated by a hinge region. It predominant role is to positively regulate the stability and translation of target mRNAs through binding to ARE (AU-rich elements) in the 3' UTR (untranslated region) of protein coding transcripts. In response to certain stimuli, HuR is subject to post-translational modifications and changes subcellular localization, which impacts its regulatory capacity. In this study on a transcriptome-wide level, we interrogate the RNA targets, precise binding sites, as well as the remodeling of these interactions in response to stimuli.</p><p>We utilized two complementary methods, RIP-chip and PAR-CLIP, to identify targets of HuR and high-resolution binding sites on a transcriptome-wide scale. We discovered that HuR-mRNA interactions are not restricted to the 3' UTR and there are thousands of intronic binding sites. A significant proportion of intronic binding sites are contained in the poly-pyrimidine tract near 3' splice sites. Binding sites in the 3' UTR and intron are often approximately 30 nucleotides apart. HuR can bind to both AU-rich and U-rich sequences, the former more prevalent in 3' UTRs and the latter more prevalent at the 3' splice site.</p><p>Next we integrated the binding data with transcriptomics of HuR siRNA mediated knockdown. We found that the degree of binding is proportional to the degree of HuR-dependent stabilization. Moreover the ability to stabilize mRNA is not restricted to 3' UTR binding sites, as intronic binding sites also exhibited the binding degree correlated stabilization. We observed that the spatial pattern of HuR binding sites relative to exons influences exon usage decisions. Specifically, binding sites upstream of the exon promote exclusion, while binding sites downstream of the exon promote inclusion.</p> / Dissertation

MicroRNAs are a large family of 21-26 nucleotide non-coding RNAs with a role in the post-transcriptional regulation of gene expression. In recent years, microRNAs have been proposed to play a significant role in the expansion of organism complexity. MicroRNAs are expressed in a cell or tissue-specific manner during embryonic development, suggesting a role in cellular differentiation. For example, Let-7 is a metazoan microRNA that acts as developmental timer between larval stages in C. elegans. We conducted a comparative study that determined the distribution of microRNA families among metazoans, including the identification of new family members for several species. MicroRNA families appear to have evolved in bursts of evolution that correlate with the advent of major metazoan groups such as vertebrates, eutherians, primates and hominids. Most microRNA families identified in these organisms appeared with or after the advent of vertebrates. Only a few of them appear to be shared between vertebrates and invertebrates. The distribution of these microRNA families supports the idea that at least one whole genome duplication event (WGS) predates the advent of vertebrates. Gene ontology analyses of the genes these microRNA families regulate show enrichments for functions related to cell differentiation and morphogenesis. MicroRNA genes appear to be under great selective constraints. Identification of conserved regions by comparative genomics allows for the computational identification of microRNAs. We have identified and characterized ultraconserved regions between the genomes of the honey bee (Apis mellifera) and the parasitic wasp (Nasonia vitripennis), and developed a strategy for the identification of microRNAs based on regions of ultraconservation. Ultraconserved regions preferentially localize within introns and intergenic regions, and are enriched in functions related to neural development. Introns harboring ultraconserved elements appear to be under negative selection and under a level of constraint that is higher than in their exonic counterparts. This level of constraint suggests functional roles yet to be discovered and suggests that introns are major players in the regulation of biological processes. Our computational strategy was able to identify new microRNA genes shared between honey bee and wasp. We recovered 41 of 45 previously validated microRNAs for these organisms, and we identified several new ones. A significant fraction of these microRNA candidates are located in introns and intergenic regions and are organized in genomic clusters. Expression of 13 of these new candidates was verified by 454 sequencing.

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.

No description available.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the expression of mRNA targets, and are aberrantly expressed in several cancers, including breast cancer. Using a transgenic mouse model of mammary tumorigenesis (MTB-IGFIR), a miRNA array was previously performed in our lab to study the expression level of various miRNAs in mammary tumours compared to wild-type mammary tissue. Next, the expression of a number of the differentially expressed miRNAs was confirmed and manipulated in a tumour cell line (RM11A) generated from a MTB-IGFIR mammary tumour. Synthetic miRNA precursors and inhibitors were then used to overexpress and knockdown, respectively, the levels of five miRNAs: miR-31, miR-183, miR-200c, miR-210, and miR-378. Upon optimization of miRNA overexpression and downregulation in RM11A cells, this study tested the effects of these miRNAs on cellular growth, survival, or invasiveness in vitro. Compared to negative controls, overexpression of all five miRNAs was associated with a significant decrease in cellular invasion, while only the overexpression of miR-31 had a significant effect on proliferation. No significant effects were found on cell survival. Our results implicate these five miRNAs in different aspects of mammary tumorigenesis, as well as having a tissue specific role in RM11A cells.

Development in multicellular organisms requires a strict balance between cell division and differentiation. The simple architecture of the Arabidopsis thaliana root makes it an ideal model for studying molecular mechanisms controlling both the transition from cell division to cell differentiation and cell fate determination. The class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factors (TFs) are well known developmental regulators, controlling important aspects of embryogenesis, shoot meristem activity, leaf polarity and vascular patterning. The HD-ZIP III TFs are under post-transcriptional control of microRNA165 (miR165) and miR166. In this thesis, I present a cell-to-cell signalling pathway underlying root vascular patterning and describe signaling pathways downstream of the HD-ZIP III TFs in their control of root development. The TF SHORTROOT (SHR), moves from the vascular stele cells to the surrounding endodermal cell layer. We show that SHR acts here to transcriptionally activate MIR165A and MIR166B, and the miR165/6 produced in the endodermis act non-cell autonomously to post-transcriptionally restrict HD-ZIP III mRNA levels in the peripheral stele. The resulting graded HD-ZIP III activity domain in the radial stele dose-dependently determines vascular cell type; high levels of HD-ZIP III in the central stele result in metaxylem formation while lower levels in the peripheral stele result in protoxylem. We provide evidence that the HD-ZIP III factors act as de novo xylem specifiers, because the quintuple mutant lacking all five HD-ZIP III genes forms no xylem. Furthermore, reducing the plasmodesmatal aperture through callose accumulation inhibits the bi-directional mobility of both signalling molecules, providing evidence that both SHR and miR165/6 move cell-to-cell via plasmodesmata to control root development. I present downstream components of the miR165/HD-ZIP III TFs in the root meristem, identified through a time-course induction of miR165 coupled to transcriptome analyses. This experiment revealed novel roles for HD-ZIP III TFs in vascular patterning and meristem size control. I show that HD-ZIP III directed repression of auxin hormone signalling in the xylem axis is essential for proper xylem differentiation. Furthermore, I provide data to show that they also control the balance of reactive oxygen species in the root meristem, thereby directing meristem size and ultimately controlling root growth.

Submitted by LÍVIA CARVALHO FERREIRA null (livinha_ferreira@hotmail.com) on 2017-12-07T17:35:22Z No. of bitstreams: 1 TESE FINAL.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2017-12-07T18:59:29Z (GMT) No. of bitstreams: 1 ferreira_lc_dr_sjrp.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) / Made available in DSpace on 2017-12-07T18:59:29Z (GMT). No. of bitstreams: 1 ferreira_lc_dr_sjrp.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) Previous issue date: 2017-11-10 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer de mama apresenta altas taxas de incidência e também de mortalidade, sendo a neoplasia mais comum entre as mulheres. MicroRNAs (miRNAs) são pequenas moléculas de RNAs não codificantes que desempenham papel fundamental na regulação gênica. Estudos recentes têm demonstrado que miRNAs estão diretamente envolvidos na iniciação e progressão de vários tipos tumorais, incluindo o câncer de mama. Diversos miRNAs têm sido descritos como promotores ou supressores tumorais, podendo estar associados ao crescimento do tumor e metástase. Atualmente, tem sido demonstrado que a administração exógena da melatonina, um hormônio naturalmente secretado pela glândula pineal, apresenta diversos efeitos oncostáticos em diferentes tipos tumorais. Assim, o objetivo do presente estudo foi avaliar o papel da melatonina em uma possível via metastática envolvendo a regulação de miRNAs em células da linhagem de câncer de mama metastática e triplo-negativa MDA-MB-231. Inicialmente, a expressão de 384 miRNAs foi avaliada utilizando placas “Taqman Low-density Array” (TLDA). Para futuras validações, foram selecionados apenas miRNAs que apresentaram fold change >1,5 e <0,5. Os resultados demonstraram que a melatonina modulou a expressão de 17 miRNAs (11 superexpressos e 6 inibidos). Dentre os miRNAs modulados pela melatonina, miR-148b e miR-210 foram confirmados por qRT-PCR, selecionados e utilizados para investigações funcionais. As células MDAMB-231 foram então transfectadas para inibição de miR-148b e/ou miR-210 (permanente ou transiente, respectivamente), e avaliadas após tratamento com melatonina. Em seguida foram avaliadas a taxa de migração e expressão proteica de cMyc, possível gene alvo de miR-148b e miR-210. Os resultados demonstraram que a melatonina foi capaz de reduzir a expressão de c-Myc e afetar a taxa de migração das células modificadas ou não com miR-148b. No entanto, nenhum efeito sobre c-Myc ou migração foi observado em células modificadas para miR-148b, quando comparadas as células controle. No que se refere ao miR-210, a melatonina alterou a expressão de cMyc e migração celular, nas células tratadas com melatonina ou expressando anti-miR210. Em resumo, nossos resultados demonstraram que apesar da melatonina influenciar na modulação positiva do miR-148b e miR-210 em células tumorais de mama triplonegativas, esta regulação não foi necessariamente causativa para alterar a expressão de c-Myc ou diminuir a taxa de migração destas células. Nossos resultados confirmam o efeito da melatonina na inibição da proliferação e migração celular, especialmente em células triplo-negativas, sugerindo seu importante papel no controle da progressão tumoral. No entanto, ainda é necessário estabelecer uma ligação direta entre as modulações de certas proteínas ou miRNAs com os efeitos da melatonina. / Breast cancer has high rates of incidence and mortality, and it is the most common cancer among women. MicroRNAs (miRNAs) are small molecules of non-coding mRNA that play a key role in gene regulation. Recent studies have shown that miRNAs are directly involved in the initiation and progression of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors of metastasis and may be associated with tumor growth and metastasis. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. Herein, we investigated if the antimetastatic effects of melatonin were coordinated by miRNAs involved in tumor progression. The expression of 384 miRNAs was measured using Taqman Low-density Array (TLDA) cards. Considering the cut-off we imposed (fold change >1.5 and (fold change >1.5 and<0.5) were evidenced the modulation of 17 miRNAs (11 up and 6 down). Among all miRNAs modulated, the selected miR-210 and miR-148b were further confirmed by qRT-PCR and tested for functional investigations. First, we engineered cells for miR-210 or miR-148b overexpression or depletion (stable or transient), then we evaluated the effect of melatonin on c-Myc protein expression and migration. Melatonin reduced c-Myc expression and migration in cell depleted or not for miR-148b. However no effect on c-Myc or migration was observed for cells depleted for miR-148b when compared with control cells. c-Myc and migration were reduced in cells treated with melatonin or expressing anti-miR-210 (depleted). In summary, our results suggest that, even if melatonin alters miRNA expression, the modulations of the miRNAs we studied, miR-210 and miR-148b, are not essential for melatonin inhibition of cell migration and c-Myc expression. One of the hypotheses is that c-Myc is not a target of these miRNAs in MDA-MB-231 cells, and it is believed that melatonin effects on miRNAs could be just “epiphenomenon” due to general Dicer/Drosha deregulations. Nevertheless, melatonin remains a powerful molecule for metastatic traits inhibition, which modulates a set of protein-coding genes, such as c-Myc. However, a direct link between expression modulations of certain proteins or miRNAs and melatonin effects has still to be established. / 2013/24612-5

Tetraploid cells, which typically arise from errors in mitosis, are genomically unstable and promote tumorigenesis. Recent evidence suggests that ~40% of tumors undergo a tetraploid intermediate during their evolution, with ~20% of all solid tumors maintaining a tetraploid karyotype. Consequently, tumor suppression mechanisms have evolved to limit the proliferation of tetraploid cells. However, it remains unclear how tetraploid cells are able to overcome these tumor suppression mechanisms to initiate tumorigenesis. To address this unresolved question, we developed and validated a genome-wide screening assay to comprehensively identify miRNAs whose overexpression promotes tetraploid cell proliferation. We then profiled those miRNAs to mechanistically define how each miRNA functions to overcome tetraploid induced arrest. Our results demonstrate that miRNAs can promote proliferation via multiple mechanisms, including inhibition of the p53 tumor suppressor pathway, hyperactivation of growth factor signaling, and inactivation of the Hippo tumor suppressor pathway. Additionally, we investigated mechanisms that facilitate tumorigenesis from proliferating tetraploid cells. It is well established that tetraploid cell proliferation promotes both numerical and structural chromosome abnormalities, although the precise mechanisms underlying these phenomena remain incompletely understood. Chromosome missegregation can lead to the formation of micronuclei separate from the primary nucleus, a result of either lagging or polar chromosomes. Micronuclei have been shown to rupture during interphase, leading to massive amounts of DNA damage and chromothripsis, resulting in extensive DNA breaks and rearrangements. We followed micronuclei formed from both lagging and polar chromosomes to determine whether all micronuclei are equally prone to nuclear envelope rupture. Our results show that polar micronuclei have nuclear envelopes that are significantly more stable than the nuclear envelopes of micronuclei formed from lagging chromosomes. Furthermore, micronuclei have been shown to be deficient at nuclear import of proteins. Kinetochore assembly, vital for proper chromosome segregation, is dependent upon the nuclear import of many proteins. We sought to establish whether micronuclei have defects in kinetochore assembly since without functional kinetochores, chromosomes cannot bind to the mitotic spindle. We found that chromosomes in micronuclei fail to assemble kinetochores efficiently, and thus promote additional chromosome missegregation. Overall, this dissertation identifies multiple mechanisms that facilitate tumorigenesis from tetraploid intermediates.

Thanks to the prove of connection between the aberrant occurrence of small RNA and various diseases and their potential in diagnostics and treatment led to discovery of new methods and materials facilitating their detection and targeted transport during gene therapy. This work summarizes present knowledge about chosen groups of small RNA, their significance in medical science and the possibilities of their detection. This work primarily concentrates on combination of magnetic separation with electrochemical detection. Magnetic particles (MPs) with different surface modifications were used for isolation. Non-specific isolation was carried out using silanol-coated MPs; streptavidin-coated MPs modified with specific biotinylated probe were used for specific separation. Square wave voltammetry (SWV) was used as a very sensitive electrochemical detection method. Optimized method based on specific magnetic separation with SWV was able to reach nanomolar detection limit (4 nM) with microRNA. The method was applied on human embryonic cells for specific isolation and detection of miR-124. The CdTe quantum dots (QDs) were studied as a nanomaterial tool for nucleic acid detection. The QDs were modified with streptavidin for their bioconjugation with biotinylated molecules were used. Interaction of QDs with nucleic acids was studied using capillary electrophoresis.