An unusual behavior of anaphors is to occur in embedded subject positions and bebound across a finite clause boundary by a matrix subject. This thesis, however, demonstrates that such constructions exist in Malki Arabic, besides other languages. First, this thesis shows that the clause size of the embedded clause in which subject anaphors are allowed is CP and not always a TP. Second, in light of current reductionist approaches to binding domains of the classical binding theory to phase theory, a cross-clausal binding relation bears issues to those approaches, as a long-distance antecedence relation crosses a phase boundary. Taking long-distance bound subject anaphors as the main empirical focus in this thesis, I show that the cross-clausal binding relation in Malki Arabic is not bona fide evidence against reducing binding domains to phases. Following Wurmbrand (2019) and Lohninger et al. (2022), I propose that constructions with long-distance bound subject anaphors theoretically resemble cross-clausal A-dependencies, like hyperraising and long-distance agreement, for undergoing movement to a position in the edge of the embedded clause and showing similar properties. Third, I show that reducing binding domains to whole phases is plausible, but taking spell-out domains as binding domains is untenable. Finally, the proposal suggested in this thesis also sheds lights on the possibility of the anaphor agreement effect as an interface condition, in addition to highlighting an account for the accusative-marked embedded subject in Modern Standard Arabic.

Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses? In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.

<p>Translesion synthesis (TLS) is a specialized type of DNA repair for bypassing DNA damage at the stalled replication fork. Because the TLS polymerases (mainly from the Y-family of polymerases) are characterized by more open active sites in order to accommodate the lesions, they are inherently more mutagenic than the replicative polymerases. Although essential for cell survival and in tolerating DNA damage, the access of the TLS polymerases to the replication fork must be tightly controlled. This regulation occurs in part through the ubiquitination state of the processivity factor PCNA. Damage-induced monoubiquitination of PCNA serves in part as the regulatory switch between replicative and translesion polymerases. Highly conserved ubiquitin-binding domains, the ubiquitin-binding zinc finger (UBZ) domain and the ubiquitin-binding motif (UBM), within the C-termini of the Y-family polymerases provide for an increased affinity of the polymerases to PCNA after damage to promote TLS. In order to determine the molecular basis for ubiquitin recognition by the TLS polymerases, we solved the solution structures of the human pol &#951; UBZ domain, the human pol &#953; UBM2 domain, and the human pol &#953; UBM2-ubiquitin complex. </p><p>The UBZ domain adopts a classical C2H2 zinc finger structure characterized by a &#946;&#946;&#945; fold, similar to the DNA-binding zinc finger proteins. Nuclear magnetic resonance titration mapped the binding interfaces between UBZ and ubiquitin to the &#945;-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single &#945;-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol &#951; UBZ domain represents a novel member of the C2H2 zinc finger family that interacts with ubiquitin to regulate translesion synthesis. </p><p>In contrast to the human UBZ domain, the yeast UBZ domain lacks one of the conserved cysteines necessary for zinc coordination, leading many to propose that it is a "zincless" zinc finger. We used biophysical methods to characterize the UBZ domains from human and yeast pol &#951; and to highlight differences between their structures and modes of ubiquitin binding. Like the human UBZ domain, the yeast UBZ domain binds zinc, which contributes to its secondary structure formation. In contrast to the human UBZ domain, the yeast UBZ domain binds to ubiquitin in a zinc-independent manner. Correspondingly, mutations in the zinc-coordinating residues of the yeast UBZ domain do not impair the polymerase's response to DNA damage.</p><p>We also investigated the structural elements and mechanism of ubiquitin recognition of the ubiquitin-binding motif (UBM) found in pol &#953; and Rev1. The solution structures of the C-terminal UBM of human pol &#953; and its complex with ubiquitin were solved. The UBM is a novel ubiquitin-binding domain that binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A of ubiquitin abolishes UBM binding. Human pol &#953; contains two functional UBMs, both of which contribute to replication foci formation. In contrast, only the second UBM of <italic>Saccharomyces cerevisiae</italic> Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations impairing the UBM-ubiquitin interaction also disrupt foci formation of pol &#953; and the DNA damage response of Rev1 <italic>in vivo</italic>, showing the significant role for the UBM in regulating TLS.</p><p>The structures of the UBZ domain and the UBM and their recognition of ubiquitin are different and distinct from other ubiquitin-binding domains. Their highly specific and unique associations with ubiquitin are critical for TLS regulation and further add to the diverse base of ubiquitin-binding domains and their role in mediating cellular functions.</p> / Dissertation

The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>