Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 211-229).

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 83-89).

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

<p>In this thesis project housekeeping genes in differentiating human embryonic stem cells were investigated. Housekeeping genes are involved in basic functions in the cells and are assumed to be expressed at relatively constant levels across different cell types and experimental conditions. Based on these features, housekeeping genes are frequently used as controls in calibration of gene expression data. Commonly used housekeeping genes in somatic tissues have shown to vary notably in human embryonic stem cells and are therefore inappropriate as reference genes in this unique cell type. In the present work a novel set of gene expression data obtained by profiling of undifferentiated and early differentiating cardiac cells, was analyzed. Stably expressed genes were identified in this data set and were subsequently intersected with a previously proposed set of 292 stable genes in human embryonic stem cells. A resulting set of 73 genes show stability across all investigated cell lines and experimental conditions. These genes are suggested as a more reliable set of reference genes in differentiating human embryonic stem cells than frequently used housekeeping genes in somatic tissue. In addition, a novel set of 20 genes was identified as very stably expressed during the differentiation towards the cardiac lineage. After further validation of stability with RT-PCR, these genes could be useful as controls in studies of human embryonic stem cells that differentiate towards the cardiac lineage.</p>

Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2008. / Guldberg, Robert, Committee Chair ; Boyan, Barbara, Committee Member ; O'Keefe, Regis, Committee Member ; Vito, Ray, Committee Member ; Bellankonda, Ravi, Committee Member.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

The family with sequence similarity 65, member B (Fam65b) protein is thought to facilitate fusion of myocytes and formation of myotubes during the differentiation of human myogenic cells. Fam65b and histone deacetylase 6 (HDAC6) co-immunoprecipitate and together regulate the levels of acetylated tubulin, which might control microtubule stability in myogenic cells. In this thesis, to gain further insight on the role of Fam65b in the differentiation pathway and motility of myogenic cells, we characterized a Fam65b knock-out (KO) mouse model. Genotyping and transcriptional analysis revealed that a thirteen exons-long region of the Fam65b gene has been successfully ablated and the mRNA amplicons within the deleted segment are not transcribed. Nevertheless, mRNA products corresponding to genomic regions downstream of the deleted area are still detected. Furthermore, analysis of skeletal muscle lysates via western blot (WB) does not show a complete loss of Fam65b expression, but only reduced translation of some isoforms. Nevertheless, WBs of myogenic cells that have been directly isolated from Fam65b KO mice and expanded in vitro revealed the absence of a 120 Kd band, which putatively corresponds to the long isoform of Fam65b. Finally, our data show that Fam65b KO mice are significantly heavier than wild type (WT) mice, and that this phenotype is consistently observed across both genders during the first seven months of age. While functional and molecular analyses of the KO mouse model are still ongoing, future work might include generating a new KO model via the CRISPR-Cas9 technology to ablate all isoforms of Fam65b.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement therapies may hold the potential for repair in a degenerate retina, by reinstating light sensitive cells to project and form connections with downstream retinal cells and finally the visual cortex. Patient-specific induced pluripotent stem cells (iPSc) could provide an autologous source of cells to replace lost tissue. However, the use of patient-derived iPSc would require that the disease-causing gene mutation be corrected in cells before transplantation. Ex vivo gene therapy of mouse photoreceptor precursor (PhRP) cells and subretinal transplantation of treated cells are here studied in a disease-specific animal model of RP; rhodopsin was ectopically expressed ex vivo in rod precursor cells, sourced from a transgenic model lacking the rhodopsin gene. Treated rod precursors were here transplanted in mice of the same disease model and are shown to gain expression of rhodopsin and mature to regenerate the absent outer nuclear layer (ONL) of degenerate mice. Visual function, assayed in the same animals before and after transplantation, was restored in animals which had no rod function at baseline. Delivery of the rhodopsin gene by both an adeno associated viral (AAV) vector and a non-viral minicircle DNA vector developed here for ex vivo gene delivery to rod photoreceptor precursors showed comparable efficiency and sustained expression. The non-viral minicircle method provides a novel system for efficient photoreceptor therapy and may offer a platform of genetic treatment of photoreceptor degenerations in which the gene in focus exceeds the size limit for packaging in AAV. Human embryonic stem cell (ESC) and human iPSC-derived PhRPs were also transplanted in mice with complete ONL degeneration and were able to reform the lost photoreceptor layer and mature in the host retina. Human cells developed light-sensitive outer segments, and reconnect with host neurons downstream to improve vision in previously blind mice. Efficient transplantation of ex vivo genetically treated rod precursors and human stem cell-derived PhRPs in animal models of progressive RP may provide a clinically-relevant model for the investigation of cell-replacement therapy for photoreceptor regeneration in retinal disease.

Orientador: Noeme Sousa Rocha / Resumo: O tumor venéreo transmissível (TVT) canino é uma neoplasia transplantável, considerada um alo-enxerto. Entretanto, pouco se sabe a respeito da origem e processo de cacinogênese. Atualmente, postula-se que alguns tumores originem-se de células iniciadoras tumorais, classicamente descritas nas leucemias mielóide humanas. As características intrínsecas do TVT fornecem indícios de uma possível participação de células iniciadores tumorais no processo de carcinogênese nesse tumor. Foi realizado estudo de fenotipagem do TVT canino para avaliar a marcação das proteínas CD44, CD133, CD90 e CD34, comumente associadas ao potencial iniciador tumoral. Para tanto utilizou-se a citometria de fluxo, imuno-histoquímica e RTq-PCR. Foram analisados também as frações de crescimento pelo Ki-67) e o número de células em apoptose pela caspase-3 clivada. Trinta e oito amostras de TVT foram obtidas de pacientes sem tratamento quimioterápico prévio. As amostras foram classificadas em plasmocitóide ou mistas, de acordo com o subtipo citológico; as células positivas na citometria de fluxo foram representadas em termos percentuais para os marcadores CD44, CD34, CD90 e CD133; a fração de crescimento foi representada pela técnica do H-Score; a quantidade de células apoptóticas foi representada pelo somatório de células positivas para a caspase-3 clivada; as imuno-marcações das proteínas CD44 e CD34 foram representadas por escores semi-quantitativos baseados na intensidade e percentual de células positivas;... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The canine transmissible venereal tumor (CTVT) is a transplantable neoplasia considered an allograft. Information about the origin and carcinogenesis process is scarcely known. Currently, some neoplasms are believed to arise from a tumor-initiating cell (TIC's) classically described in human myeloid leukemia. TVT intrinsic characteristics provide evidence of a possible TIC's participation in carcinogenesis process of this malignancy. Thus, a phenotyping study of CTVT was conducted to assess the immunophenotyping properties of the proteins CD44, CD133, CD90 and CD34, already known to be associated to tumor initiator potential. The use of flow cytometry and immunohistochemistry contributed to this purpose. In addition, growth fractions and cells undergoing apoptosis were examined by Ki-67 and caspase-3 cleaved, respectively. Thirty-eight samples were chosen from patients having no previous chemotherapy and cytological diagnosis of CTVT. Samples were classified into plasmacytoid or mixed according to cytological subtype. Positive cells in the flow cytometry were expressed in percentage for the markers CD44, CD34, CD90 and CD133. H-score technique helped to represent growth fractions. Apoptotic cell quantity was calculated by summing positive cells. Immunohistochemical marking of CD44 and CD34 proteins were determined by semiquantitative scores based on the intensity and percentage of positive cells. Moreover, specimens were divided into resistant and non-resistant groups and com... (Complete abstract click electronic access below) / Doutor

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), whose function is to optimise neuronal transmission and preserving axonal integrity. Oligodendrocytes are derived from a stem cell population, called oligodendrocyte progenitor cells (OPCs). Oligodendrocyte lineage cells (OLCs) have been implicated in the pathophysiology of various diseases including not only demyelinating diseases (eg. Multiple Sclerosis (MS) or Pelizaeus-Merzbacher disease (PMD)), but also psychiatric disorders (eg. schizophrenia or Rett syndrome (RTT)). Regardless of the type of disease, understanding the underlying fundamental biology of the oligodendrocyte lineage cells is pivotal to develop therapeutic strategies. In the mouse embryonic forebrain OPCs are generated in consecutive waves from distinct brain regions along a spatiotemporal gradient; with ventral OPCs emerging before dorsal OPCs. The developmentally distinct OPCs, and their progenies, persist in the brain throughout life. To investigate whether ventrally and dorsally derived OLCs fulfil different functions in the adult brain, dorsally derived OPCs were ablated in development using a \textit{Sox10}-driven diphtheria toxin fragment A (DTA) mouse model. As dorsally derived OPCs populate the cortex, locomotor coordination and cognition were investigated following dorsal OPC ablation. Mice ablated of the dorsal OPC population do not show a significant deficit in learning and attentional function. In contrast, ablated mice show an impaired locomotor coordination, while general vigilance, gait, balance and sensation are comparable to control groups. The locomotor coordination disabilities are a result of alterations of brain, not spinal cord homeostasis, as only a neglect able number of OLCs in the spinal cord are affected by the ablation model. In addition, no signs of neuronal cell death or chronic inflammatory response was detected in response to the ablation. As the oligodendrocyte numbers are similar between control and ablated animals, the locomotor coordination phenotype can also not be explained by reduced number of oligodendrocytes. However, clustering analysis following single-cell Drop-sequencing uncovered a heterogeneity of oligodendrocyte (OL) subpopulations in the motor cortex. Whilst some OL subpopulations are of mixed developmental origin, others are exclusively formed by either ventrally or dorsally derived OLs, arguing that dorsal oligodendrocyte subpopulations are crucial for homeostatic brain function. In the absence of dorsal OPCs, ventral OPCs are not capable of forming dorsal oligodendrocyte subpopulations in response to dorsal OPC ablation. In conclusion, my results indicate a functional heterogeneity of developmentally-distinct oligodendrocytes in physiological brain function.