Pax genes encode for transcription factors essential for tissue development in many species. Pax8, the only member of the family expressed in the thyroid tissue, is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-specific genes. TTF-1, a homeodomain-containing factor, is also expressed in the thyroid tissue and has been demonstrated to play a role in thyroid-specific gene expression. Despite the presence of Pax8 and TTF-1 also in a few other tissues, the simultaneous expression of the two transcription factors occurs only in the thyroid, supporting the idea that Pax8 and TTF-1 might cooperate to influence thyroid-specific gene expression. In this report, we describe a physical and functional interaction between these two factors. The fusion protein GST-Pax8 is able to bind TTF-1 present in thyroid or in non-thyroid cell extracts, and by using bacterial purified TTF-1 we demonstrate that the interaction is direct. By co-immunoprecipitation, we also show that the interaction between the two proteins occurs in vivo in thyroid cells. Tissue-specific transcriptional regulation is often mediated by a complex of cis-acting elements. The vast majority of the promoters of genes expressed in a cell type-specific fashion contains a variety of recognition sequences for tissue-specific and ubiquitous transcription factors. It is well known that tissue-specific transcriptional regulation is mediated by a set of transcription factors whose combination is unique to the cell type. Thyroid follicular cells, the most abundant cell population of the thyroid gland, represent a useful model system to elucidate the mechanism operating in the establishment and maintenance of cell type-specific expression. Thyrocytes are responsible for thyroid hormone synthesis and are characterized by the expression of a specific set of genes such as thyroglobulin (Tg) 1 and thyroperoxidase (TPO), which are exclusively expressed in this cell type (1, 2), and by the expression of genes expressed only in a few tissues other than the thyroid, such as the thyrotropin-stimulating hormone receptor and the sodium/ iodide symporter.The Tg and TPO promoters have been extensively studied, and multiple factors have been shown to be required for their expression (3, 4). To date, three transcription factors that specifically bind to and regulate these promoters have been cloned (2). The three transcription factors are as follows: thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2), and Pax8. TTF-1 (also named NKx 2.1 and T/EBP) is a homeodomain-containing protein expressed in embryonic diencephalon, thyroid, and lung (5). TTF-2 is a forkhead domaincontaining protein expressed in pituitary and thyroid (6), and Pax8 is a member of the murine Pax family of paired domaincontaining genes that is expressed in kidney, in the developing excretory system, and in the thyroid (7). We have focused our studies on the molecular mechanisms of action of TTF-1 and Pax8. These two transcription factors are pre...

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The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein-protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

Nowadays, Quality Management tools such as failure mode and effect analysis (FMEA) are widely used throughout the aeronautical, automotive, software, food services, health care and many other industries to sustain and improve quality and safety. The increasing complexity of scientific research makes it more difficult to maintain all activities under control, in order to guarantee validity and reproducibility of results. Even in non-regulated research, scientists need to be supported with management tools that maximize study performance and outcomes, while facilitating the research process. Frequently, steps that involve human intervention are the weak links in the process. Risk analysis therefore gives considerable benefit to analytical validation, assessing and avoiding failures due to human error, potential imprecision in applying protocols, uncertainty in equipment function and imperfect control of materials. This paper describes in detail how FMEA methodology can be applied as a performance improvement tool in the field of non-regulated research, specifically on a basic Life Sciences research process. We chose as "pilot process" the selection of oligonucleotide aptamers for therapeutic purposes, as an example of a complex and multi-step process, suitable for technology transfer. We applied FMEA methodology, seeking every opportunity for error and its impact on process output, and then, a set of improvement actions was generated covering most aspects of laboratory practice, such as equipment management and staff training. We also propose a useful tool supporting the risk assessment of research processes and its outputs and that we named "FMEA strip worksheet." These tools can help scientists working in non-regulated research to approach Quality Management and to perform risk evaluation of key scientific procedures and processes with the final aim to increase and better control efficiency and efficacy of their research.