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This thesis titled, “Nature of Local Interactions at cisPro-Aro Peptide Sequences in Proteins: Evidences for van der Waals type Interactions. Design and Synthesis of Novel Covalent Surrogates for the Peptide Hydrogen Bond”, describes two important studies. The first is to gain a thorough understanding of the nature of interactions that govern cisPro stability at Pro-Aro sequences, which described in the first four chapters. The final chapter describes the synthesis of novel 4-carbon covalent surrogates for the peptide H-bonding interaction. Chapter 1: Local Interactions Governing cisPro Stability: Refining the Model Peptides Chapter 1 Section A: Understanding the role of inter-side chain CH•••Aro interaction in cis-trans isomerization at Pro-Aro and Aro-Pro Sequences. This chapter is divided into two sections. In the first section an exhaustive overview of earlier investigations into the nature of local interactions at Xaa-cisPro-Aro and Aro-cisPro-Xaa peptide sequences, by various groups, are discussed. Most studies have found evidence for the close assemblage between side chains of residues flanking cisPro motifs, when at least one of them is an aromatic group. An electronic C-H•••π nature has been proposed for these assemblies and they are proposed to influence the cisPro stability. We highlight those features in these studies that indicate that these interactions are not always electronically tunable, are insensitive to presence of strong chaotropes in the solvent and occur at protein sequences lacking Pro or cisPro; all of which contradict the electronic C-H•••π model for these inter-side chain assemblages and their perceived influence on cisPro stability. Chapter 1 Section B: Investigation of the Nature of H Xaa•••Aro interaction at Xaa-Pro-Pro-Phe-sequences In Section B, we design and synthesize Pro-Aro containing short peptide models to investigate the nature of local C-H•••Aro interactions in them. We synthesize a series of homologous Pro-Pro-Aro containing peptides (modeled based on earlier studies) and investigate the relative populations of its four Xaa-Pro rotamers using extensive 1D and 2D NMR techniques including TOCSY, HSQC and ROESY. We find several drawbacks that make this a relatively deficient model. Firstly, their relative populations of the rotamers (the most important data for current investigation) cannot be determined with high fidelity as they are dependent on the solvent polarity, solute concentration and chemical shift degeneracy of crucial NMR signals for the rotamers. Importantly, the populations of a few rotamers are influenced by strong 13-membered ring backbone H-bonds. Notably, some of the cisPro rotamers do not even contain the inter-side chain assembly, whose nature is under investigation. Design of novel models – unconstrained by H-bonds We design the Acyl-Pro-Pro-Aro-OMe peptides that lack the possibility of forming the 13-membered ring H-bonded structures. Thorough 1D and 2D NMR analyses of these models reveal that strong Type VI β-turn type 10-membered ring H-bonds are formed in the rotamers of these models – hence precluding their applications for current study. Interestingly, the relative rotamer populations are strongly influenced by solvent polarity and are entirely different from those of the corresponding C-terminal amide models. We further discover that the Pro-Pro-Aro motif is not essential to express the inter-side chain interactions – Ala-Pro-Aro are sufficient. Formation of the 10-membered H-bonding interactions, however, are not precluded. Chapter 2: Design and Synthesis of Acyl-Pro-Phe-OMe: Novel models to investigate the role of HαXaa•••Aro interactions on Xaa-cisPro-Aro stability. Chapter 2 Section A: Design, Synthesis and Conformational Analysis of Ibu-Pro-Phe-OMe Chapter 2 is divided into two sections. In Section A, we replace the amino acid at the N-terminal of the putative Pro residue with simple isosteric isobutyryl group, the resulting minimalist dipeptide model shows the exclusive influence of desired inter-side chain interactions in the cisPro rotamer. Solvent polarity and temperature coefficient studies reveal that absence of any intramolecular H-bonding or Oπ* interactions in it. 1D and 2D NMR analyses clearly indicate the close proximity between the side chains of Ibu and Phe exclusively in the cisPro rotamer. The Kc/t value decreases upon mutation of Phe to Ala. All these features favor the Ibu-Pro-Phe-OMe as an ideal minimalistic model for investigating the nature of Ibu•••Ph assemblages in the cisPro rotamer. Chapter 2 Section B: Investigation of CH•••Aro /Alp•••Alp interactions in Ibu-cisPro-Xaa-OMe In Section B, the 1D and 2D NMR analyses of the complete set of the aliphatic and aromatic analogues Ibu-Pro-Xaa-OMe were investigated. DMSO-d6 was found to be the best solvent for mimicking both the folded and the unfolded local environments of these short peptide sequences. The HαIbu•••Aro assemblage is observed in Aro analogues, but cannot be electronically tuned. The aliphatic analogues also surprisingly contain the HαIbu•••Alp interactions! The Kc/t values (cisPro %) increase in the aliphatic analogues too, where the aliphatic side chain is long. Increase in cisPro stability is not due to ring current effects or intramolecular H-bonds or Oπ* interactions. It seems to be due to van der Waals type interactions between the involved side chains, either of which need not be aromatic in nature. Chapter 3: Nature of Inter-Side Chain Interactions at Acyl-cisPro-Aro Sequences: Evidence for van der Waals Interactions Chapter 3 Section A: Investigation of nature of inter-side chain interactions in R-CO-cisPro-Phe-OMe Chapter 3 has two sections. Section A describes the systematic design and synthesis of Acyl-Pro-Phe-OMe homologues where first the steric bulk and hence the surface area of the aliphatic side chain of the acyl group is varied. Interaction of the phenyl ring of Phe seems to occur with the Cα-Cβ σ-bond of the acyl group. Branching at either Cα or Cβ seems to destabilize the cisPro rotamer. Aliphatic•••Aromatic interactions overwhelm the cisPro rotamer population to be greater than that of transPro. In the analogues where the acidity of the acyl Cα-H bond is increased, the Kc/t does not increase correspondingly. The Δδ(trans-cis) ppm shifts of HαAcyl protons are dependent exclusively on its acidity rather than on the Kc/t values. In carbamyl-Pro, which entirely lack the HαAcyl proton, the Kc/t values are significantly high and improve as the aliphatic surface on the alkoxy group increases. Introduction of benzyloxy carbamyl group at Pro renders almost the same Kc/t values as that of ethyloxy carbamate. All these data contradict the C-H•••π interaction model and strongly support a van der Waals type interaction between the Acyl (preceding cisPro) group’s Xα-Yβ σ-bond and the Aro or Alp side chains (succeeding cisPro). Chapter 3 Section B: Evidence for the Van der Waals nature of Inter Side Chain (Acyl•••S.C.Aro/Alp) interactions- Determination of Interactions energies In Section B, a thorough investigation of both aliphatic•••aliphatic and aliphatic•••aromatic interactions on the background of homologous Acyl-Pro-Aro/Alp-OMe peptide models is undertaken. These models uniquely allow the delineation of contribution of the van der Waals interactions and the ring current effects to the cis/trans isomerization in these peptides. We see that the energy of the van der Waals component of these aliphatic•••aliphatic and aliphatic…aromatic interactions increase linearly with increase in Kc/t, in both DMSO-d6 and D2O. On other hand, energy from the ring current effects largely remains invariant. The Acyl•••Aro/Alp interactions are not hydrophobic and are facilitated by conformational effects. Chapter 4: Crystallographic evidence for van der Waals interaction-mediated stabilization of cisPro conformers Chapter 4 Section A: Systematic crystallization and crystal structure analyses of homologous Xaa-cisPro-Alp and Xaa-cisPro-Aro rotamers: Evidence for van der Waals interactions Chapter 4 has two sections, both of which present crystallographic evidence for the van der Waals nature of the Xaa•••Aro interactions at Xaa-cisPro-Aro sequences. Section A describes the unique crystal structures of five of the Acyl-Pro-Alp-OMe analogues that have been synthesized in the current study. All of them remarkably crystallize with two features: 1) the Acyl-Pro peptide bond adopts the cisPro rotamer in all; and 2) the aliphatic side chains of the acyl group and the Alp side chain are involved in van der Waals type interactions. The cisPro rotamers of even the bulkiest motifs, namely Ibu-Pro-Val-OMe, Piv-Pro-Ile-OMe and Piv-Pro-Leu-OMe crystallize, stabilized by van der Waals packing between aliphatic groups of the acyl and the Leu/Ile/Val side chains. Where the side chains are not long enough to make sub-van der Waals contacts with each other, their acyl C′-Cα σ-bond rotations are restricted due to Oσ* interactions involving the charge on the acyl carbonyl O. Where this occurs, the short space between the acyl and Alp side chains are filled in by aliphatic groups from neighbouring molecules at sub van der Waals distances. The Pro, Alp and χ1(Alp) dihedral angles are restricted to narrow range of values, irrespective of the length of Alp side chain, indicating that this backbone conformation is a conformational minimum when i+3i backbone H-bond is removed, with Pro at the i+1st position. This is further substantiated in Piv-Pro-Gly-OMe, which crystallizes in trans-Pro form, but still adopts similar backbone dihedral angles in spite of lacking any Alp side chain for interactions with the acyl group. Three of the Acyl-Pro-Aro-OMe models also crystallize in cisPro rotamer forms – both exhibit van der Waals type contacts between the Acyl group and backbone of Phe, rather than the aromatic ring of Phe. The phenyl ring of Phe may or may not form intramolecular Ph•••Pro inter-side chain contacts – which is not a pre-requisite for cisPro stabilization. No C-H••• interactions are observed anywhere in these peptides – van der Waals type contacts alone predominate in all cases. There are no abnormal distortions in bond angles or lengths even in the most sterically hindered cases, signifying that the conformations of these cisPro rotamers involving aliphatic•••aliphatic type contacts are natural minima. Chapter 4 Section B: Mining the PDB for Statistical Evidence of van der Waals interactions Section B of chapter 4 describes the data mining and statistical analyses of Xaa-cisPro-Phe, Xaa-cisPro-Val and Xaa-cisProLeu sequences in the PDB. The PEARL program was used to mine the PDB data. The overall frequency of 5.3% for appearance of cisPro among all Xaa-Pro peptide bonds, improves when Xaa is Phe or Tyr. However, several anomalies highlight the need for refining the analyses set to only those sequences where the side chains of Xaa and Aro/Alp face each other. In this refined set, clearly, inter side chain Xaa•••Alp/Aro contacts take precedence over even Aro•••Pro interactions at Aro-cisPro sequences (where Xaa is Aro). The Phe and the Leu side chains induce similar conformational effects on the preceding Xaa-Pro backbone. So does Val. Strong aliphatic•••aliphatic inter side chain contacts at van der Waals distances are observed to flank cisPro in several proteins. Substitution at the Cα of Xaa governs the proximity of the approaching side chain of Alp / Aro residue. The Cα-H of Xaa steers away from the Aro side chain at Xaa-Pro-Phe sequences, as the Aro group gets closer to it – implying the absence of ordered C-H••• contacts between them. There is consistent parallel alignment between Cα-Cβ -bond of Xaa and the C -C bond of the approaching side chain of Alp or Aro group – clearly highlighting the presence of van der Waals type interactions between them. All these evidences clearly point towards the van der Waals nature of local interactions at cisPro-Aro/Alp peptide sequences. Chapter 5: A novel 4-carbon covalent surrogate model for peptide H-Bond Chapter 5 describes the design and synthesis of novel 4-carbon covalent surrogates for the peptide H-bond (HBS). These surrogates would allow the unique constraining of two peptide strands in their extended conformations. The covalent HBS contain four orthogonal functional groups for independent extension at all of the four ends – similar to an endogenous inter-strand peptide H-bond. The synthesis of the surrogate is achieved by directly using natural chiral amino acid derivatives, beginning from amino alcohols obtained from reduction of desired amino acids. Suitably N-protected alcohols undergo oxidation to aldehyde followed by Grignard addition of allyl magnesium bromide, TBDMS protection of the homoallylic alcohol and reductive ozonolysis of the olefin to get a primary alcohol which is subject to Fukuyama-Mitsunobu reaction with desire protected peptide. The residue preferences that produce strongest inter-strand H-bonds were explored. The designed 4-carbon covalent HBS was incorporated using this methodology in a Gramicidin-S analogue, its first structural mimic containing only a single turn motif. This HBS model will have wide applications for constraining peptides in a number of secondary structures.

Arkadia participa do processo de amplificação da sinalização de TGF-b mediada por Smads, via degradação do I-Smad. O objetivo desse estudo foi caracterizar e investigar a influência de Arkadia em linhagens celulares de cânceres de tiróide. A expressão gênica de Arkadia em linhagens celulares de carcinomas papílifero (NPA), folicular (WRO) e anaplásico (ARO), foi avaliada por PCR quantitativo. Em ARO, que apresenta a maior expressão de Arkadia, foram identificados subclones (ARO_1 e ARO_2) com expressão diferencial de Arkadia, ARO_2>ARO_1. A expressão gênica de SMAD2, 3, 4, 7 e de genes do ciclo celular modulados por TGF-b, foi maior em ARO_2. Os subclones respondem ao tratamento com peptídeo de TGF-b1 e activina A. O crescimento in vivo (xenotransplante) mostra que ARO_2 desenvolve um tumor de menor volume. Recentemente a origem de ARO foi questionada e comprovamos sua origem por análises de expressão gênica e morfologias. Desta maneira, observamos que a expressão diferencial de Arkadia indica que ela está envolvida na modulação inibitória da via de TGF-b. / Arkadia is involved in the process of amplification of the TGF-b signaling mediated by Smads, by degradation of I-Smad. The aim of this study was to characterize and investigate the influence of Arkadia in thyroid cancers cell lines. Arkadia gene expression in the papillary (NPA), follicular (WRO) and anaplastic carcinoma cell lines (ARO) was evaluated by quantitative PCR. In ARO, which presents the highest Arkadia expression, we identified subclones (ARO_1 and ARO_2) with differential Arkadia expression ARO_2> ARO_1. The expression of SMAD2, 3, 4, 7 and the cell cycle genes modulated by TGF-b, was also higher in ARO_2. However both the subclones responded to treatment with peptide of TGF-b1 and activin A. The in vivo growth (evaluated by xenotransplant), showed that ARO_2 developed tumors of lower volume. Recently the ARO origin was questioned and we proved its origin by gene expression and morphological analysis. This way, the differential Arkadia expression indicates that it is involved in modulation of the inhibitory TGF-b pathway.

Arkadia participa do processo de amplificação da sinalização de TGF-b mediada por Smads, via degradação do I-Smad. O objetivo desse estudo foi caracterizar e investigar a influência de Arkadia em linhagens celulares de cânceres de tiróide. A expressão gênica de Arkadia em linhagens celulares de carcinomas papílifero (NPA), folicular (WRO) e anaplásico (ARO), foi avaliada por PCR quantitativo. Em ARO, que apresenta a maior expressão de Arkadia, foram identificados subclones (ARO_1 e ARO_2) com expressão diferencial de Arkadia, ARO_2>ARO_1. A expressão gênica de SMAD2, 3, 4, 7 e de genes do ciclo celular modulados por TGF-b, foi maior em ARO_2. Os subclones respondem ao tratamento com peptídeo de TGF-b1 e activina A. O crescimento in vivo (xenotransplante) mostra que ARO_2 desenvolve um tumor de menor volume. Recentemente a origem de ARO foi questionada e comprovamos sua origem por análises de expressão gênica e morfologias. Desta maneira, observamos que a expressão diferencial de Arkadia indica que ela está envolvida na modulação inibitória da via de TGF-b. / Arkadia is involved in the process of amplification of the TGF-b signaling mediated by Smads, by degradation of I-Smad. The aim of this study was to characterize and investigate the influence of Arkadia in thyroid cancers cell lines. Arkadia gene expression in the papillary (NPA), follicular (WRO) and anaplastic carcinoma cell lines (ARO) was evaluated by quantitative PCR. In ARO, which presents the highest Arkadia expression, we identified subclones (ARO_1 and ARO_2) with differential Arkadia expression ARO_2> ARO_1. The expression of SMAD2, 3, 4, 7 and the cell cycle genes modulated by TGF-b, was also higher in ARO_2. However both the subclones responded to treatment with peptide of TGF-b1 and activin A. The in vivo growth (evaluated by xenotransplant), showed that ARO_2 developed tumors of lower volume. Recently the ARO origin was questioned and we proved its origin by gene expression and morphological analysis. This way, the differential Arkadia expression indicates that it is involved in modulation of the inhibitory TGF-b pathway.

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The thesis focuses on analysis and design of a special hospital bed designed for the department of anesthesiology and resuscitation. The design was based on the rules set by the ČSN EN 60601-2-52 norm. The bed is adjusted for the use at the department of anesthesiology and resuscitation not only with its look but also with its functions. The design emphasizes the ergonomics of the bed, safety and the needs of the patient as well as of the staff and also an easy maintenance of the bed. Some of the main innovative elements are: basal stimulation system placed directly in the bed, used colors, more infusion-rack holders or a headboard fixed to the construction of the bed if not needed. The bed was designed especially to provide more comfort to the patient.

Summary: The diploma thesis compares competency of nurses working in intensive care in The Czech Republic and Great Britain. Theoretical part of the thesis gives compact look to regulation of nursing profession, education of nurses and nursing registration in both countries. In this part are highlighted main differences of the legislation which nursing competency is based on in each country. There is also mentioned the public opinion of the nursing as a profession and its prestige related to both countries. Practical part of the thesis focuses on the interpretation of analysis of the research regarding the above mentioned topic. For the research have been used the method of collecting data by structured questionnaires. There was a questionnaire for each country in its own language. The aim of the practical part is to compare divergence of knowledge, skills and competency of nurses working in intensive care in both countries. Alternatively to assess any reason for exceeding nursing competency and to find out the explanation for that Key Words: Competency, Nurse, Critical Care, Education, Qualification, Great Britain

The thesis titled Securing airways in pediatric patients on ARD deals with the specifics of nursing care of airways. Care of airways of children hospitalised at the Anaesthetic Resuscitation Department is an integral part of comprehensive nursing care. Each nurse must have a sufficient theoretical and practical knowledge, as well as experience to independently perform nursing care of airways of pediatric patients. Free and patent airway is a prerequisite for ensuring one of the basic life functions. The aim of the theoretical part of the thesis was to summarise the issue of securing airway in children and to focus on nursing care for airways in pediatric patients. The aim of the research was to determine the specifics of nursing care for children hospitalised at the Anaesthetic Resuscitation Department due to impaired breathing. Based on the aim of the thesis, the author set out four research questions: 1. What nursing activities are performed by nurses when securing an airway in a child? 2. How nurses treat airways of intubated children? 3. What nursing care do nurses provide for children before, during and after extubation? 4. What nursing care do nurses provide for children with a tracheostomy? In the empirical part of the thesis the author employed qualitative research. Data collection was performed by the use of individual interviews with nurses. The survey was conducted at a pediatric ICU and a children's Anaesthetic Resuscitation Department. Based on the research questions and information obtained from literature sources the author drew up the basic points for interviews with nurses. The interviews were supplemented by an observation performed on a children's Anaesthetic Resuscitation Department. Interviews and observation were complemented with two case reports of pediatric patients. The reports were processed in accordance with Henderson's model. The thesis's results show specifics and difficulties of nursing care of pediatric patients hospitalised at an Anaesthetic Resuscitation Department. The thesis maps nursing care for pediatric patients when securing airway, it focuses on intubation, tracheostomy tube, extubation, aspiration, and care for oral and nasal cavities. The objective of the thesis is to show not only the specifics of this kind of nursing care and also to demonstrate the importance of communication and emotional support to pediatric patients before, during, and after the medical intervention. The intensive nursing care is continuously improving thanks to new methods and devices. The results will be provided to the nursing management of the pediatric ICU and the children's Anaesthetic Resuscitation Department. They will also be presented at professional seminars and in professional journals.

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