<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

Many of today’s methods for the detection of biomolecules suffer from a high limit ofdetection due to poor signal generation upon recognition of target. By applying andoptimizing proximity ligation assay (PLA) in Western blotting (WB), the limit of detectionhas been lowered down to the picomolar range. In this report I have optimized the differentparameters that affect the signal generation and explored possibilities to increase the ease ofuse, by merging protocol steps and performing signal generating reactions at roomtemperature.

<p>with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome. The mechanisms of this enhanced susceptibility remain unknown. It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation. Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis. The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown. Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation. First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery. Five rats were used as the controls. Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats (p=0.002). Immuno-electron microscopy confirmed localization of ED-1 in PIMs. BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017) but not of IL-1â, TNF-á, TGF-â and IL-10. Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-â and IL-10.</p><p>I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous). All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment. Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000). Staining of lung sections with an LPS antibody localized the LPS in lungs. RT-PCR analyses showed no differences in IL-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats. Immuno-electron microscopy showed IL-1â and TNF-á in PIMs. BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000 & 0.002 respectively) in lungs compared to LPS challenged control rats. TGF-â mRNA did not change (p=0.128) but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS. </p><p> To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge. The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours. Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group). There were no differences in IL-1â mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712 & 0.509 respectively). ELISA showed no decrease in IL-1â concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455 & 0.008 respectively). TNF-á mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499 & 0.297 for 6 hour & 48 hour GC groups respectively). But TNF-á concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group. IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038 & 0.000 respectively) compared to LPS challenged BDL rats. ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030) but not in 6 hour GC group (p=0.420). TGF-â mRNA expression was decreased in 48 hour GC group (p=0.000) but not in 6 hour GC group (p=0.182). But GC treatment did not affect TGF-â concentrations. </p><p>The data from these experiments characterize a BDL model to study PIM biology, show PIMs pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.</p>

Lifes origin is, in many ways, coupled to understanding the evolution of nucleic acids. In contemporary life, proteins and nucleic acids are intricately dependent upon each other for a host of functions including, but not limited to, replication and chemical ligation. Protein enzymes are necessary for the synthesis of DNA and RNA, while nucleic acids are necessary for both the coding and synthesis of proteins. According to the RNA World hypothesis, early life used nucleic acids for both information storage and chemical catalysis before the emergence of protein enzymes. However, it still remains a mystery how nucleic acids were able to assemble and replicate before the advent of protein enzymes. We have utilized the ability of small molecule intercalation to assemble nucleic acids into stable secondary structures. Our motivation in this pursuit comes from the recently proposed Molecular Midwife hypothesis where small molecules may have acted as nanoscale structural scaffolds upon which the nucleic acid bases were able to stack into stable structures and undergo assembly into polymers. We have also found that the kinetics and thermodynamics of small molecule-mediated assembly and secondary structure formation are strongly dependent upon oligonucleotide length. Small molecules bind to nucleic acids by multiple modes of binding and this phenomenon must be properly understood in order to achieve robust and versatile assembly of nucleic acid structures.

M. tuberculosis copper-sensitive operon repressor (Mtb CsoR) is the founding member of a new metalloregulatory protein family in prokaryotes that regulates the transcription of the cso operon in response to copper toxicity. Mtb CsoR tetramer binds 1 monomer mol equiv of Cu(I) with very high affinity (log KCu=18.0) via three conserved residues, Cys36, His61' and Cys65'. Binding of Cu(I) allosterically inhibits the CsoR binding to the DNA operator (CsoO) overlapping the cso promoter (DeltaGc=+3.6 kcal/mol, pH 7.0, 25 oC). These findings are consistent with a role of CsoR as a transcriptional repressor with Cu(I) binding inducing transcriptional derepression. To explore the mechanism of this regulation, His61 was substituted with 1-methylhistidine (MeH) or Beta- (2-thiazolyl)-alanine (Thz) using a native chemical ligation strategy. The CsoO binding affinities of the resultant H61MeH and H61Thz CsoRs are both refractory to inhibition by Cu(I) binding despite the fact that each forms a high affinity 3-coordinate complex with Cu(I). This suggests that while Cu(I) is coordinated by the N?11 atom of His61, the N?22 atom plays an critical role in driving this allosteric switch. Evidence in support of a formation of a hydrogen bonding network involving the N?1 face of His61 and two conserved "second coordination shell" residues, Glu81' and Tyr35, is presented. Remarkably, this mechanism is analogous to that proposed for the Zn(II) sensor CzrA from S. aureus. To test this, we employed the same native chemical ligation approach to substitute the key Zn(II) ligand His97 with 1-methylhistidine; with the preliminary findings fully consistent with an intersubunit allosteric switch involving the N?2 face of this key His97 residue in CzrA. Two predicted homologs of Mtb CsoR were also biochemically characterized to obtain additional support for the hypothesis that CsoR is a key Cu(I) regulatory protein in many bacterial species. B. subtilis CsoR, known to regulate the transcription of the copZA operon, was found to have biochemical properties similar to those of Mtb CsoR as to Cu(I) binding, DNA binding and Cu(I)-dependent allosteric regulation. Interestingly, Bsu CsoR also binds other divalent metal ions (Zn, Ni) with high affinity but with metal coordination geometries distinct from that of Cu(I). Binding of these divalent metal ions only weakly regulates copZA operator binding in vitro, suggesting that coordination number and geometry are most closely related to the allosteric regulation. Finally, a putative CsoR from the pathogenic S. aureus Newman strain was identified and characterized, and was found to exhibit biochemical properties similar to those of Mtb and Bsu CsoRs. Parallels between Cu(I)-sensing CsoRs and functional orthologs in the CsoR/RcnR family are further discussed in the context of the mechanism and evolutionary divergence of this new family of regulatory proteins.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

No description available.

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.

The aim of this study was to investigate the viability of laparoscopic testicular artery ligation as an alternative to the more conventional castration methods in equids. Twelve intact male donkeys varying in age from 5 months to 13 years were used in this study. Two of these donkeys were castrated by means of an open technique. The remaining donkeys underwent testicular artery ligation under laparoscopic guidance. Ligation of the testicular artery was achieved by application of Filshie clips under laparoscopic visualisation. This was performed with the donkeys standing in stocks and sedated with a combination of detomidine hydrochloride and butorphanol tartrate. The ten donkeys which underwent testicular artery ligation were unilaterally castrated using an acceptable open castration technique on two separate occasions. The first testes were removed at a specific time period after testicular artery ligation, while the second testes were all removed on the same day 9 months after the first group of donkeys underwent laparoscopic assisted testicular artery ligation. The remaining two donkeys which acted as controls underwent a standard open bilateral castration procedure both testes being removed on the same day. All the testes removed, excluding one testis lost to the study, were evaluated histologically. Four testes were used as controls. Thirteen testes showed histological signs of ischaemic necrosis, degeneration and/or atrophy. Three testes showed histological tubular degeneration and atrophy as well as the presence of some normal tissue. The remaining three testes were histologically unchanged. One of the testes was accidentally misplaced at post mortem due to human error. Avascular necrosis of the testicular tissue was not successful in all the subjects. This technique shows promise but further research is required. / Dissertation (MMedVet)--University of Pretoria, 2008. / Companion Animal Clinical Studies / unrestricted

Les dendrimères greffés de L-lysine (DGL) sont des matériaux aux applications potentielles variées (agents antibactériens, antifongiques, ?). Ces dendrimères greffés sont obtenus selon un procédé original mis au point au laboratoire, basé sur la polymérisation des N-carboxyanhydrides (NCA) de Llysine. Le travail effectué au cours de cette thèse a pour objectif d'explorer les différents modes de modification des DGL et d'en évaluer la faisabilité. Pour ce faire, dans une première partie, nous avons détaillé le processus de synthèse et de polymérisation en milieu hétérogène des NCA. Cela nous a permis, ensuite, en modulant la nature de l'agent d'amorçage de la polymérisation des NCA, d'examiner la possibilité d'introduire de nouvelles fonctionnalités (fluorophore, bras de liaison adapté à la ligation) sur la première génération de DGL. Cela a nécessité, en amont, la synthèse d'un bras espaceur hétérobifonctionnel de type polyéthylène glycol. Par la suite, une ligation de type oxime entre les nouveaux DGL de 1ère et de 2e génération et une molécule modèle a été réalisée afin de valider cette approche. Enfin, nous avons pu démontrer que ces dendrimères greffés pouvaient être fonctionnalisés en surface par divers substrats (sucre, guanidine, acide aspartique, acide succinique) leur conférant de nouvelles propriétés et ouvrant ainsi un vaste champ d'application pour ces macromolécules. / L-lysine Dendrigraft (DGL) are materials with various potential applications (antibacterial, antifungal, ...). These dendrigrafts are obtained by an original process, developed in our laboratory, based on the polymerization of N-carboxyanhydrides (NCA) of L-lysine. The work carried out during this thesis was aimed at exploring different methods of modification of DGL and assessing their feasibility. In the first part, we detailed the process of synthesis and polymerization of NCAs in a heterogeneous medium. This allowed us then by modulating the initiator of NCA polymerization to introduce new features (fluorophore, spacer adapted to the ligation) on the first generation of DGL. This required, upstream, the synthesis of a heterobifunctional polyethylene glycol type spacer. Subsequently, an oxime ligation between the new 1st and 2nd generation of DGL and an aldehyde model has been conducted to validate this approach. Finally, we demonstrated that these dendrigrafts could be functionalized on the surface by various substrates (sugar, guanidine, aspartic acid, succinic acid) giving them new properties and opening up a wide scope of applications for these macromolecules.