<p>Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. <i>In situ</i> hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.</p>

<p>All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. </p><p>The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. </p><p>The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes.</p><p>In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; <i>MZF6D</i>, <i>HKr18</i>, <i>HKr19</i> and <i>HZF12</i>. Whereas <i>HKr18</i> and <i>HZF12</i> seem to be ubiquitously expressed, <i>MZF6D</i> and <i>HKr19</i> show a more restricted expression pattern. Northern blot and <i>in situ</i> hybridisation analyses of <i>MZF6D</i> showed that the expression of this gene is restricted to meiotic germ cells. <i>MZF6D</i> might thus be involved in the formation of male gametes. The expression of <i>HKr19</i>, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.</p>

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 138-153).

The gluing of unseasoned wood, called green gluing, is a relatively new sawmill process, implying a radically changed order of material flow in the production of value-added wood-based products. It facilitates the enhancement of raw material recovery and value yield by integrating defect elimination and gluing already before kiln drying. The present study evaluates green glued adhesive bonds in flatwise glued beams and finger joints. The main part of this work deals with green gluing using a moisture curing polyurethane adhesive (PUR). Standardised test methods and specially designed, small scale, specimens were used for the determination of the strength, fracture energy and the ductility of both dry- and green glued bonds in tension and in shear. Using the small scale specimens it was possible to capture the complete stress versus deformation curves, including also their unloading part. An optical system for deformation measurement was used for the analysis of bond behaviour. The influence of moisture content during curing and temperature after curing on the adhesive chemical composition and on the mechanical properties was investigated. Furthermore, the moisture transport through the adhesive bond during curing was tested. Finally, microscopy studies were performed for analysis of bond morphology and fracture. The results show that two significant factors influence the shear strength of green glued bonds: wood density and adhesive spread rate. Bonds which fulfil the requirements according to EN 386 could be obtained within a wide range of process parameters. The small specimen tests showed that green glued PUR bonds can reach the same strength and fracture energy, both in shear and in tension, as dry glued bonds with the same adhesive amount. The local material properties of the bonds could be determined, thanks to the failure in the tests taking place within the adhesive bond itself and not in the wood. Following process factors were shown to cause lower bond strength: a) a low adhesive spread rate, b) high pressure and c) short pressing time in combination with low wood density and high moisture content. Moreover, the heat treatment of the cured PUR adhesive during drying influenced the chemical composition of the adhesive, providing for higher strength, stiffness and Tg of the adhesive, caused by an increased amount of highly ordered bidentate urea.

Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. In situ hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.

All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes. In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; MZF6D, HKr18, HKr19 and HZF12. Whereas HKr18 and HZF12 seem to be ubiquitously expressed, MZF6D and HKr19 show a more restricted expression pattern. Northern blot and in situ hybridisation analyses of MZF6D showed that the expression of this gene is restricted to meiotic germ cells. MZF6D might thus be involved in the formation of male gametes. The expression of HKr19, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.

The fidelity of an organism’s genome is central to biology. DNA, however, is constantly being damaged and modified by a variety of sources. As a result of these changes, repair enzymes, polymerases, and other interrogating biomolecules must be able to recognize, repair, and adapt to a multitude of different structures and dynamics presented. Manipulation of natural systems via the development and introduction of novel bases and DNA structures only adds to this complexity. In addition, specific RNA sequences are becoming more prevalent therapeutic and diagnostic targets. These include retroviruses and other viruses that maintain their genome with RNA. Unlike DNA, RNA poses a unique challenge as targets due to their highly diverse secondary and tertiary structures. In this manuscript, three different nucleic acid systems were chosen to investigate how intramolecular and intermolecular interactions impact their own structure as well as giving further insight into how nucleic acids are recognized and distorted by interrogating damage specific enzymes as well as structure specific proteins.

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