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No / Following legislative changes in 2004 and the establishment of the Human Tissue Authority, access to human tissues for biomedical research became a more onerous and tightly regulated process. Ethical Tissue was established to meet the growing demand for human tissues, using a process that provided ease of access by researchers whilst maintaining the highest ethical and regulatory standards. The establishment of a licensed research tissue bank entailed several key criteria covering ethical, legal, financial and logistical issues being met. A wide range of stakeholders, including the HTA, University of Bradford, flagged LREC, hospital trusts and clinical groups were also integral to the process.

Yes / For dental human enamel, what is the precise mineralization progression spatially and the precise timings of mineralization? This is an important question in the fundamental understanding of matrix-mediated biomineralization events, but in particular because we can use our understanding of this natural tissue growth in humans to develop biomimetic approaches to repair and replace lost enamel tissue. It is important to understand human tissues in particular since different species have quite distinct spatial and temporal progression of mineralization. In this study, five human central incisors at different stages of enamel maturation/mineralization were spatially mapped using synchrotron X-ray diffraction and X-ray microtomography techniques. From the earliest developmental stage, two crystallite-orientation populations coexist with angular separations between the crystallite populations averaging approximately 40o and varying as a function of position with the tooth crown. In general, population one had significantly lower texture magnitude and contributed a higher percentage to the overall crystalline structure, compared to population two which only contributed 20-30% but had significantly higher texture magnitude. This quantitative analysis allows us to understand the complex and co-operative structure-function relationship between two populations of crystallites within human enamel. There was an increase in the mineral concentration from the enamel-dentin junction peripherally and from the incisal tip cervically as a function of maturation time. Quantitative backscattered-electron analyses revealed that mineralization of prism cores precedes that of prism boundaries. These results provide new insights into the precise understanding of the natural growth of human enamel. / Partly funded by NERC grant ”Timelines in Teeth” NE/F018096/2.

Background: 1985 saw the beginnings of a population-based biobank in Västerbotten County, Sweden. In 1999, a start-up genomics company, UmanGenomics, obtained ‘all commercial rights’ to the biobank. The company introduced an ethics policy, which was well received in prestigious journals, focusing on public oversight and informed consent. Aims: To explore how social anthropology can aid understanding of the challenges posed by the new role of the biobank in Västerbotten, and thus complement more established traditions in the field of medical ethics. An anthropological study of the ethics policy was executed. Theoretical perspective: Inspired by the anthropology of policy and social science perspectives on ethics and morality, the policy was studied at three analytical levels: policymakers (who formulate the policy), policy workers (who implement the policy, primarily nurses who obtain informed consent) and target group (for whom and on whom the policy is supposed to work: the potential donors to the biobank). Methods: Policymakers, nurses, and potential donors were interviewed, donations observed, and official documents analysed to mirror the moral problematizations made at the three levels in each other and to study the practical implications of the policy. To extend the reliability of the findings two surveys were executed: one among the general population, one among donors. Results: The qualitative studies show that policymakers distinguish between blood and data differently to potential donors. Informed consent seems more important to policymakers than potential donors, who are more concerned about political implications at a societal level. Among the respondents from the survey in the general public, a majority (66.8%) accepted surrogate decisions by Research Ethics Committees; a minority (4 %) stated informed consent as a principal concern; and genetic research based on biobank material was generally accepted (71%). Among the respondents to the survey in donors, 65% knew they had consented to donate a blood sample, and 32% knew they could withdraw their consent; 6% were dissatisfied with the information they had received; and 85% accepted surrogate decisions by Research Ethics Committees. Discussion: The ethics policy constitutes a particular naming and framing of moral problems in biobank-based research which overemphasises the need for informed consent, and underemphasises other concerns of potential donors. This embodies a political transformation where access to stored blood and medical information is negotiated in ethical terms, while it also has unacknowledged political implications. In particular, the relations between authorities and citizens in the Swedish welfare state are apparently transforming: from mutual obligation to individual contracts. Conclusion: Anthropology contributes to medical ethics with increased awareness of the practical implications of particular research ethical initiatives. This awareness promotes appreciation of the political implications of ethics policies and raises new issues for further consideration.

Orientador: Prof. Dr. Luciano Puzer / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017. / As calicreinas 5 (KLK5) e 7 (KLK7) sao serino proteases que pertencem a um grupo de 15 calicrei.nas teciduais humanas (KLKs) encontradas em diversos tecidos do corpo. A KLK5 e encontrada principalmente no tecido mamario, sistema nervoso central, testiculos, prostata e traqueia, enquanto a KLK7 e encontrada no esofago, figado, rins e glandulas salivares. Alem disso, ambas as enzimas sao mais abundantemente expressas na pele humana, onde acredita-se que exercam importante papel no processo de descamacao epidermal, a partir da hidrolise de proteinas que compoem as estruturas de adesao intercelular dos corneodesmossomos. Estudos tambem demonstram, que a atividade dessas duas enzimas aparece aumentada em patologias relacionadas ao processo de descamacao da pele, como psoriase e dermatite atopica. Desta forma, o desenvolvimento de inibidores para KLK5 e KLK7 podera contribuir nao apenas para elucidar seus mecanismos fisiologicos e patologicos, mas como um arquetipo de novas drogas, visando ao desenvolvimento de novos procedimentos terapeuticos para patologias relacionadas ao processo de descamacao epidermal. Neste projeto, foram testados 17 pseudopeptideos e 10 compostos derivados do acido tetramico frente as calicrei.nas teciduais humanas 5 e 7, tripsina e quimotripsina. Dos 17 pseudopeptideos, oito compostos nao foram soluveis na fase de teste em meio aquoso e nove compostos foram soluveis em meio aquoso e puderam ser testados como inibidores das KLK 5 e KLK 7, sendo que quatro destes apresentaram inibicao utilizando concentracoes da ordem micromolar perante a KLK5. No entanto, apenas um composto obteve acao inibitoria frente a KLK7, porem com potencial inibitorio menor, comparado com resultados anteriores, relatados pelo nosso grupo de pesquisa. Os dez compostos aciltetramicos foram soluveis em tampao aquoso e apenas tres nao apresentaram atividade inibitoria contra as enzimas. Os resultados referentes as KLKs e tripsina foram significativos com uma acao inibitoria da ordem de micromolar. Os compostos derivados do acido tetramico nao inibiram a quimotripsina. Esses resultados abrem uma discussao sobre a especificidade da KLK7, que e classificada como uma enzima do tipo quimotripsina-like, no entanto, neste trabalho, apresentou um padrao de inibicao semelhante a tripsina. / The kallikreins 5 and 7 are serine proteases which belong to a group of fifteen human tissue kallikreins (KLKs) found in a diverse of body tissues. The KLK5 is mainly found on mammary tissue, central nervous system, testicles, prostate and trachea, while the KLK7 is found in the esophagus, liver, kidneys and salivary glands. Moreover, both enzymes are abundantly expressed on the human skin, and its believed that they have an important role on the epidermal desquamation process, from the hydrolysis of proteins that make up the corneodesmosomes intercellular adhesion structures. Recently it was shown that both kallikreins activity appears to be increased in diseases relates to the desquamation process, such as psoriasis and atopic dermatitis. Thus, the development of inhibitors for KLK5 and KLK7 would not only contribute to the elucidation of their physiological and pathological mechanisms, but also serve as an archetype for new drugs, aiming the development of new therapeutic procedures for diseases related to epidermal desquamation. In this project, 17 pseudo-peptides and 10 tetramic acid derived compounds were tested against the human tissue kallikreins 5 and 7, trypsin and chymotrypsin. From the 17 pseudo-peptides, nine were soluble in aqueous medium and could be tested; four of them showed inhibition in the micro molar range against the KLK5. Just one compound was effective against the KLK7, but with low inhibitory action. The 10 aciltetramic compounds were soluble in aqueous buffer, and only three of them did not show any inhibitory activity against the kallikreins. The results referring to the kallikreins and trypsin were significant, with an inhibitory action in the micro molar range. The compounds did not inhibit the chymotrypsin. These results open a discussion about the KLK7 specificity, which despite of being classified as a chymotrypsin-like enzyme, showed, in this work, an inhibition pattern similar to trypsin.

Orientador: Prof. Dr. Luciano Puzer / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / As calicreinas teciduais humanas (KLKs) compreendem uma familia de quinze serino proteases encontradas em uma diversidade de fluidos e tecidos biologicos. Estas enzimas sao identificadas como possuindo papel em diferentes doencas como Alzheimer, cancer, dermatite atopica, esclerose multipla, Parkinson, psoriase e outras. Existe, portanto, uma crescente demanda por inibidores especificos para cada uma das calicreinas e este e o objetivo do nosso grupo de pesquisa na UFABC. Neste trabalho pretendemos gerar inibidores para as calicreinas teciduais humanas 3, 5 e 7, utilizando bibliotecas baseadas em duas serpinas diferentes: uma expressando a forma Pittsburgh do inibidor de proteinase-¿¿1 (IP-¿¿1 M358R), randomizada nos residuos 352-356 (P7-P3); e outra expressando a serpina bacteriana vioserpina, randomizada nos residuos 343-347 (P3-P2f). A abordagem do phage display foi eficaz para gerar as bibliotecas e o protocolo de bioselecao utilizado adequado para enriquecer diversas variantes reativas. Na selecao da biblioteca do IP-¿¿1 M358R, consensos PSEAL e PSRIL foram observados, para KLK5 e KLK7, respectivamente, e varias das sequencias selecionadas exibiram maiores taxas de inibicao para ambas as calicreinas, quando comparadas a molecula molde (IP-¿¿1 M358R). A variante HDVIL e o consenso PSRIL foram identificados como sendo altamente seletivos para a KLK7, com constantes de segunda ordem 14 e 33 vezes maiores que as para KLK5. Pudemos realizar uma selecao efetiva da biblioteca de vioserpina contra a KLK7, cujas variantes enriquecidos demonstraram uma preferencia geral pelo aminoacido Serina ocupando as posicoes P3, P1f, P2f e P1, seguido por uma Tirosina, tambem preferida em P2. A tecnica de phage display foi, portanto, eficiente como base para um estudo de especificidade, e para o desenvolvimento de melhores e mais especificos inibidores para as Calicreinas Teciduais Humanas, e pode ser utilizada para o desenvolvimento de novas bibliotecas, com outras regioes da RCL randomizadas, ou mesmo baseadas em outras serpinas. / The human tissue kallikreins (KLKs) comprise a family of fifteen serine proteases found in a diversity of biological fluids and tissues. These enzymes are identified as having a role in different diseases such as Alzheimer's, cancer, atopic dermatitis, multiple sclerosis, Parkinson's, psoriasis, and others. Thus there is a growing demand for specific inhibitors for each of these kallikreins, and this is the aim of our group at UFABC. In this work we intended to generate inhibitors for the human tissue kallikreins 3, 5 and 7, using libraries based on two different serpins: one expressing the Pittsburgh form of the human serpin á1-proteinase inhibitor (á1-PI M358R), randomized at residues 352-356 (P7-P3); and another one expressing the bacterial vioserpin, randomized at residues 343-347 (P3-P2¿). The phage display approach was effective to generate the libraries and the biopanning protocol used suitable to enrich numerous reactive variants. On the á1-PI M358R selection, loose consensus of PSEAL and PSRIL were observed, for KLK5 and KLK7, respectively, and several of the selected sequences exhibited higher inhibition rates when compared to the template molecule for both kallikreins. The variant HDVIL and consensus PSRIL were found to be highly selective for the KLK7, with second order constants 14- and 33-fold higher than the ones for KLK5. We could only perform an effective selection with the vioserpin library for the KLK7, whose enriched variants demonstrated a general preference for the amino acid Serine occupying the positions P3, P1¿, P2¿ and P1, followed by a Tyrosine, also preferred on the P2. The phage display approach was therefore effective as basis for a specificity study, and for the development of improved, more specific inhibitors for the Human Tissue Kallikreins, and can be used to develop new libraries, with other randomized RCL regions, or even based on other serpins.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18138号 / 医博第3858号 / 新制||医||1002(附属図書館) / 30996 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 坂田 隆造, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stem cell technologies as a branch of regenerative medicine are becoming increasingly popular as the science behind it evolves. Therefore, it is important that the regulatory framework pertaining to stem cell technologies be well defined and appropriate to prevent unethical and unscrupulous behaviour on the part of medical practitioners, which gives rise to stem cell tourism. South African legislation pertaining to stem cell technology is regarded as inadequate and dissonant with the Constitution, exacerbating the problem of stem cell tourism and denying patients access to certain stem cell therapies, which ultimately can be viewed as an infringement of their constitutional rights. The United Kingdom (UK) provides a clear-cut regulatory framework, which is not only centred around consent and patient safety but is also conducive to production of stem cell therapies. For such reasons, this dissertation finds the UK framework to be an appropriate benchmark against which the South African regulatory framework can be evaluated. By means of comparison and elaborating on the biology of stem cells in addition to pertinent ethical principles, legislation and human rights of both South Africa and the UK, an argument will be made out that South African legislation pertaining to stem cell therapy and related matters is wanting. Furthermore, analysis will be made of the definition of biological medicine as put forward by the Medicines and Related Substances Control Act 101 of 1965 to conclude that certain stem cell therapies are best excluded from such a definition as such stringent requirements and protocols encumbers access to stem cell therapies and inflates costs. Lastly, remedial measures are proposed to remedy these injustices by proposing for the institution of a specialist adivisary committee to oversee stem cell and related activities. Key Words: Regenerative Medicine; Stem Cells; Stem Cell Regulation; National Health Act; Medicines and Related Substances Control Act; Advanced Therapy Medicinal Product; Human Tissue Authority; Human Fertilisation and Embryology Authority; HTA; HFEA; Medicine and Healthcare Products Regulatory Agency; MHRA; European Medicines Agency; Tissue-engineered Products; Doctor-Patient Relationship; Medical Innovation Bill 2014; Experimental Treatments; Innovative Therapy; Hospital Exemption; Informed Consent; Special Exemption; Autologous Stem Cell Therapy; Stem Cell Transplants; Gene Therapy Advisory Committee. / Dissertation (LLM)--University of Pretoria, 2017. / Public Law / LLM / Unrestricted

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).