Biology is the study of life and life systems. Until recently, biologists have concentrated in examining how cells proliferate, differentiate, and survive in biological systems. Although such studies reveal extremely interesting insights into the complexity of living organisms, there is much more to this story. Just as cells live, cells must also eventually die. The study of how and why cells die has become the focus much scientific research over the past decade. Because the regulation of the number and specificity of cells in the immune system is critical to the life of a mammalian organism, researchers began to investigate how this strict control was accomplished. It was found that large quantities of immature T cells die in the course of their development by a specific type of cell death process coined apoptosis. This Ph.D. dissertation was directed towards examining the mechanisms involved in the regulation of thymocyte apoptosis in a murine model system. The first portion of the project involved isolating differentially regulated genes using either a plus/minus or subtractive hybridization screening strategy The second component of this dissertation investigated possible roles molecular oxygen and/or free radicals play during thymocyte apoptosis. Results from these studies both identified numerous putative death transcripts as well as revealed the requirement for oxygen during cell death in thymocytes induced by specific stimuli.

Glioblastoma, previously known as Glioblastoma Multiforme (GBM), is a highly angiogenic tumor and is defined pathologically by its ability to create microvascular proliferations. Patients with glioblastoma have a high rate of morbidity and mortality because of the aggressive nature of this type of tumor. Despite current optimal treatments, glioblastoma is almost universally fatal with the current median survival being between 12–15 months. The current treatment for glioblastoma involves surgical resection followed by chemotherapy and radiation. However, recently a new anti-angiogenic treatment called Bevacizumab (trade name Avastin) has been able to prolong survival, significantly in some cases. Some patients on this therapy have demonstrated wonderful responses, but in most cases, this response is transitory and there is eventual recurrence, thus highlighting that there could be resistance to anti-angiogenic therapies inherent within glioblastoma. Here, it is proposed that this resistance is mediated by an alternative angiogenic pathway that is produced by glioblastoma stem-like cells. Through a trans-differentiation process, we have found that the vast majority of the vasculature (80%) acquires a mural cell phenotype that "mimics" the vasculature and creates a blood supply to the tumor. Although this process has been defined in other types of cancer as Vasculogenic Mimicry, this process has never been demonstrated to be due to mural cell involvement. We have also identified and more fully characterized this alternative form of vasculogenesis by its dependence on a molecular pathway through which it is created and maintained in glioblastoma. We have found that the glioblastoma stem-like cells transdifferentiated in vitro, and in vivo in a Flk-1 (VEGFR2) dependent manner when transplanted into SCID/beige mice. When this receptor was knocked down in transdifferentiated glioblastoma stem-like cells, it inhibited tumor growth and development almost completely. Since current angiogenic therapies for glioblastoma do not target this form of vasculogenesis, it is hoped that this work will in part fill a void in the current research and create additional knowledge to help better guide treatment for these patients.

When fractionated using an HPLC ion exchange column, three distinct peaks (peak I, II, and III) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla cells, suggesting the presence of at least three different phosphatases. These phosphatases showed different activities toward phosphocasein in the presence of Mg$\sp{2+}$ and Mn$\sp{2+}$. Peak III, which represents about 50% of the active enzyme activity when phosphocasein is used as substrate, showed a molecular weight of 140 KDa as determined by HPLC gel filtration and has been identified as a type 2A protein phosphatase. Okadaic acid, a phosphatase inhibitor (specific for type 2A and type 1) and tumor promoter, was employed to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. After 3 days cultured in the presence of 50 ng/ml NGF, 20-25% of the PC12 cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC$\sb{50}$ of about 7 nM. This inhibition was rapidly reversed after wash-out of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells indicating that continual phosphatase activity is required to maintain neurites. A 27-mer oligonucleotide was synthesized as a hybridization probe to isolate clones encoding the sequence of protein phosphatases from a bovine adrenal medulla cDNA library. A cDNA clone encoding the full length of the catalytic subunit of protein phosphatase type 2A has been isolated. The deduced protein sequence (309 residues, 35.63 KDa) is 99.7% identical to that of phosphatase 2A$\alpha$ form from rabbit skeletal muscle, human liver and porcine kidney and differs by only one amino acid (Arg-55 vs. Cys-55). At the nucleotide level, the clone showed 97% identity with that of the catalytic subunit of protein phosphatase type 2A$\alpha$ from human liver. Sequence comparison of bovine adrenal medulla clone with phosphatase type 1 from rabbit skeletal muscle and type 2B from mouse brain identifies six highly conserved domains in the three enzymes that are expected to be crucial for the catalytic activity of protein phosphatase.

The intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, undergo two periods of developmentally programmed cell death. The first period occurs during the larval/pupal transition where half of the ISMs die and the remainder persist throughout pupal development until death upon eclosion of the adult. Both periods of ISM death employ much of the same molecular machinery even though the endocrine cues for the degeneration process is different for each period of muscle death. A gene that is dramatically increased in its expression during both periods of ISM death is polyubiquitin. This gene has been isolated and characterized in this research. Despite a large amount of allelic heterogeneity in the population, it has been determined that not only is this gene increased in response to developmental cues, but that its transcript is also increased in response to stress. Therefore, polyubiquitin is multifaceted in its regulation. Due to the diverse transcriptional regulation of this gene, it was determined to be a good candidate in which to develop a method to introduce promoter/reporter constructs into the ISMs of the hawkmoth. Even though this research does not conclusively demonstrate that reporter activity after transfection of the promoter/reporter constructs is due to the polyubiquitin promoter, the research does demonstrate that DNA mediated transfection of insect muscle tissue can be achieved.

<p>Over the course of development, cells and tissues of the embryo must take on the correct fates and morphologies to produce a functioning organism. The patterning events and morphogenetic processes that accomplish this task have been the subject of decades of research, the consequence of which has been a detailed comprehension of the molecular mechanisms that regulate each. Equally important is an understanding of the mechanisms that coordinate patterning with morphogenesis, such that they occur with the correct relative spatiotemporal dynamics. My thesis work sought to characterize such co-regulation in the context of two developmental events in a vertebrate model, the African clawed frog Xenopus laevis: induction of bottle cell formation at the onset of gastrulation after germ layer induction, and regulation of the morphogenetic movements of neurulation in relation to neural plate patterning. Chapter 1 of this dissertation provides a general introduction to the patterning and morphogenetic events of early development relevant to my thesis. Chapter 2 presents a discussion of my work to characterize the function of two signaling pathways, namely Nodal signaling and Wnt/Planar Cell Polarity, in the induction of bottle cells. My experiments confirm the requirement for Nodal signaling in bottle cell induction, but do not support a role for the individual transcriptional targets of Nodal signaling tested here or for Wnt/PCP. Chapter 3 summarizes my work to address the function of two transcription factor-encoding genes, sall1 and sall4, in neural development, including their roles in anteroposterior neural patterning, neural tube morphogenesis, and neural differentiation. My work shows that both sall1 and sall4 are required for all three processes, and supports the hypothesis that their key role in this context is to transcriptionally repress stem cell factors of the pou5f3 family, allowing progression through neural development. As a whole, this work summarizes my research to characterize molecules that co-regulate early patterning and morphogenetic events in the X. laevis embryo.

Long-distance signaling is an important process in the development of Arabidopsis thaliana. A leucine-rich repeat receptor-like kinase (LRR-RLK), XYLEM INTERMIXED WITH PHLOEM1 a.k.a. C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (XIP1/CEPR1), functions in vascular development and has recently been implicated in nitrogen sensing and response. Previous results indicate that XIP1/CEPR1 also interacts with multiple proteins involved in sugar metabolism and transport as well as other metabolic proteins, which indicates a possible role for XIP1/CEPR1 in mediating sugar transport. xip1-1 seeds, which grow slowly in the cold in comparison to Columbia wild-type plants, were previously EMS mutagenized and screened for suppressors of the cold-sensitive phenotype. One of these suppressors, 9-12, maps to the lower region of chromosome V and several possible causative EMS-like mutations have been identified that may link XIP1/CEPR1 to a more general vascular transport role.

Doutoramento em Ciências Farmacêuticas / PhD in Pharmaceutical Sciences

Each step of vascular development needs to be carefully regulated; endothelial precursors must be specified, these cells then proliferate and coalesce to form vascular cords, and finally they lumenate, undergo angiogenic branching and remodeling, and recruit smooth muscle cells to establish a mature vessel. An aberration at any of these steps during embryonic development is incompatible with life, and vascular pathologies in the adult are associated with numerous diseases including stroke, arteriosclerosis, diabetic retinopathies and cancer progression. My work has aimed to understand how endothelial precursors are specified, and more precisely the cell-signaling pathways and transcriptional networks that guide their fate. This work leads us to conclude the following: (1) blood island precursor cells in the Xenopus embryo can give rise to either blood or endothelial cells, and it is BMP-mediated activation of the erythroid transcriptional program that regulates cell fate, (2) endothelial specification requires the Ets transcription factor Etv2. Persistence of Etv2 expression in blood/endothelial cell precursors allows these cells to develop into endothelium, and overexpression of Etv2 in any of the three germ layers causes activation of every endothelial marker examined. Along the way we have characterized a number of small-molecule inhibitors that should be useful to the Xenopus community and applicable to other model systems.

<p> The studies described in this thesis explore the physiology of upper respiratory tract colonization by S. pneumoniae and S. pyogenes. We have shown that colonization is associated with biofilm formation and examined the effects of co-colonization on genetic exchange. Additional studies have investigated the virulence and inflammatory potential of biofilm bacteria, and identified factors influencing cellular egress from biofilm communities and the transition to acute disease. The remaining chapters explore the mechanism of action of the human milk protein lipid complex HAMLET and its potential for antimicrobial adjutancy.</p>

<p> The origins of pairs of monozygotic twins and higher order multiples, i.e. triplets, quadruplets, etc., have been extensively studied but still little is understood. To gain insight into this event, certain possible cell labeling schemes that model an organism&rsquo;s development are analyzed. The phenomenon of quadruplet twins is exposed during the process. We predict that monozygotic quadruplets are not really quadruplets but instead are two pairs of monozygotic twins where the pairs slightly differ. From the considered models, the probability of monozygotic twins is found to be (1/2)<i>^K</i>, and we discover from our analysis that the probability of monozygotic quadruplets, or triplets in the case of the death of an embryo, is (1/8)<i>^K</i>, where <i> K</i> is a species-specific integer representing the number of pairs of homologous chromosomes. This investigation into twinning provides a foundation for understanding the process of cell development through which the cell development mechanism is established. The failure of the internal cellular clock from this mechanism may play an important role in cancerogenesis. The parameter K may determine cancerization with a probability threshold that is approximately inversely proportional to the Hayflick limit, so exposure to small levels of ionizing radiation and chemical pollution may not produce cancer.</p>