Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D. / Oomycetes (water molds) are eukaryotic microorganisms that resemble filamentous fungi (molds), but are actually relatives of diatoms and brown algae, within a different kingdom of life named Stramenopila. The functional relationship between oomycetes and fungi is similar to that between fish and dolphins, which also acquired similar functions via different evolutionary paths. Many families of oomycetes are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Other families of oomycetes are animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. <i>Phytophthora</i> species, represented by the Irish Potato Famine pathogen <i>P. infestans</i> and the soybean pathogen <i>P. sojae</i>, are among the most destructive oomycete pathogens of plants and thus have been extensively studied. This dissertation is focused on the model oomycete pathogen <i>P. sojae</i>. It investigates specific aspects of its molecular biology and establishes an efficient genetic manipulation tool. All complex organisms (eukaryotes) package their genetic material in nuclei, which contain proteins as well as DNA. In the first part of my research (Chapter 2 and Chapter 3), I focused on the mechanisms used by <i>P. sojae</i> to target nuclear proteins into the nucleus, particularly the tags (called nuclear localization signals, or NLSs) that are identify the proteins that must travel to the nucleus. I showed that nuclear targeting mechanisms in oomycetes differ in distinct ways from well-studied eukaryotes such as humans. In particular, the nuclear targeting signals in <i>P. sojae</i> proteins are diffused over multiple sites on the proteins, whereas in human proteins there’s usually just a single signal. For one particular oomycete protein, a transcription factor, nuclear targeting involves four weak signals that cooperate synergistically. Two of these four weak signals define a new class of nuclear localization signal. In the second part of my research (Chapter 4 and Chapter 5), I implemented and further optimized a genome editing technology for genetic modification and control of <i>P. sojae</i>. This technology is based on the CRISPR system that has revolutionized genome editing in plants and animals over the last three years. This is of particular importance because genome editing techniques were not previously possible in oomycetes. The successful implementation of CRISPR technology in <i>P. sojae</i> has provided a major new research capability to the oomycete community. In Chapter 5, I also describe several optimized methodologies for <i>P. sojae</i> genetic manipulation based on my 5 years of experience with <i>P. sojae</i>.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 174-182). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

En aquesta tesi s'ha caracteritzat la ruta d'internalització de l'onconasa, una RNasa citotòxica. Els resultats indiquen que l'onconasa entra a les cèl·lules per la via dependent de clatrina i del complex AP-2. Seguidament es dirigeix als endosomes de reciclatge i es a través d'aquesta ruta que la proteïna exerceix la citotoxicitat. Per altra banda, els resultats d'aquest treball demostren que PE5, una variant citotòxica de la ribonucleasa pancreàtica humana (HP-RNasa), interacciona amb la importina &#61537; mitjançant diferents residus que tot i que no són seqüencials, es troben propers en l'estructura tridimensional d'aquesta proteïna. PM8 és una HP-RNasa amb estructura cristal·logràfica dimèrica constituïda per intercanvi de dominis N-terminals. En aquesta tesi s'han establert les condicions per estabilitzar aquest dimer en solució i també es proposa un mecanisme per la dimerització. / In this thesis it has been characterized the internalization pathway of onconase, which is a cytotoxic ribonuclease. The results show that onconase enters cells using AP-2/clathrin mediated pathway and then is routed to the recycling endosomes. In addition, the results show that this is the route used by onconase to perform its cytotoxicity. On the other hand, the results indicate that PE5, a cytotoxic human pancreatic ribonuclease (HP-RNase), interacts with importin &#945; using different residues that although they are scattered along the sequence, they are close in the three-dimensional structure of the protein. PM8 constitutes a crystallographic dimer by the exchange of the N-terminal domains. In this thesis it has been investigated the solution conditions that favour the dimeric form and it is proposed a dimerization process of this variant. Finally, the pattern of substrate cleavage is studied by HP-RNase.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references.

The family of runt related transcription factors (RUNX/Cbfa/AML/PEBP2) are essential for cellular differentiation and fetal development. RUNX factors are distributed throughout the nucleus in punctate foci that are associated with the nuclear matrix/scaffold and generally correspond with sites of active transcription. Truncations of RUNX proteins that eliminate the C-terminus including a 31-amino acid segment designated the nuclear matrix targeting signal (NMTS) lose nuclear matrix association and result in lethal hematopoietic (RUNX1) and skeletal (RUNX2) phenotypes in mice. These findings suggest that the targeting of RUNX factors to subnuclear foci may mediate the formation of multimeric regulatory complexes and contribute to transcriptional control. In this study, we hypothesized that RUNX transcription factors may dynamically move through the nucleus and associate with subnuclear domains in a C-terminal dependent mechanism to regulate transcription. Therefore, we investigated the subnuclear distribution and mobility of RUNX transcription factors in living cells using enhanced green fluorescent protein (EGFP) fused to RUNX proteins. The RUNX C-terminus was demonstrated to be necessary for the dynamic association of RUNX with stable subnuclear domains. Time-lapse fluorescence microscopy showed that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space in living cells. By measuring fluorescence recovery after photobleaching, both RUNX1 and RUNX2 were found to dynamically and rapidly associate with these subnuclear foci with a half-time of recovery in the ten-second time scale. A large immobile fraction of RUNX1 and RUNX2 proteins was observed in the photobleaching experiments, which suggests that this fraction of RUNX1 and RUNX2 proteins are immobilized through the C-terminal domain by interacting with the nuclear architecture. Truncation of the C-terminus of RUNX2, which removes the NMTS as well as several co-regulatory protein interaction domains, increases the mobility of RUNX2 by at least an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. Contributions of the NMTS sequence to the subnuclear distribution and mobility of RUNX2 were further assessed by creating point mutations in the NMTS of RUNX2 fused to EGFP. The results show that these point mutations decrease, but do not abolish, association with the nuclear matrix compared to wild-type EGFP-RUNX2. Three patterns of subnuclear distribution were similarly observed in living cells for both NMTS mutants and wild-type RUNX2. Furthermore, the NMTS mutations showed no measurable effect on the mobility of RUNX2. However, the mobility of RUNX proteins in each of the different subnuclear distributions observed in living cells were significantly different from each other. The punctate distribution appears to correlate with higher fluorescence intensity, suggesting that the protein concentration in the cell may have an effect on the formation or size of the foci. These findings suggest that the entire NMTS and/or the co-regulatory protein interaction domains may be necessary to immobilize RUNX2 proteins. Because RUNX factors contain a conserved intranuclear targeting signal, we examined whether RUNX1 and RUNX2 are targeted to common subnuclear domains. The results show that RUNX1 and RUNX2 colocalized in common subnuclear foci. Furthermore, RUNX subnuclear foci contain the co-regulatory protein CBFβ, which heterodimerizes with RUNX factors, and nascent transcripts as shown by BrUTP incorporation. These results suggest that RUNX subnuclear foci may represent sites of transcription containing multi-subunit transcription factor complexes. RUNX2 transcription factors induce expression of the osteocalcin promoter during osteoblast differentiation and to study both RUNX2 and osteocalcin function, it would be helpful to have transgenic mice in which OC expression could be easily evaluated. Therefore, to assess the in vivo regulation of osteocalcin by RUNX protein, we generated transgenic mice expressing EGFP controlled by the osteocalcin promoter. Our results show that EGFP is expressed from the OC promoter in a cultured osteosarcoma cell line, but not in a kidney cell line, and is induced by vitamin D3. Furthermore, the OC-EGFP transgenic mice specifically express EGFP in osteoblasts and osteocytes in bone tissues. Moreover, EGFP is expressed in mineralized bone nodules of differentiated bone marrow derived from transgenic mice. Thus, these mice produce a good model for studying the in vivo effects of RUNX-mediated osteocalcin regulation and for developing potential drug therapies for bone diseases. Taken together, our results in living cells support the conclusion that RUNX transcription factors dynamically associate with stationary subnuclear foci in a C-terminal dependent mechanism to regulate gene expression. Moreover, RUNX subnuclear foci represent transcription sites containing nascent transcripts and co-regulatory interacting proteins. These conclusions provide a mechanism for how RUNX transcription factors may associate with subnuclear foci to regulate gene expression. Furthermore, the OC-EGFP transgenic mice now provide a useful tool for studying the in vivo function and regulation of osteocalcin by RUNX proteins during osteoblast differentiation and possibly for developing therapeutic drugs for treatment of bone diseases in the future.