猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究

为解决供体器官的不足，以细胞移植为基础的替代疗法已成为治疗不可逆肝 脏疾病新的希望。 肝前体（干）细胞（Hepatic progenitor cellS，HPCs）和 胚胎干细胞（embryoic stem cells, ES）由于其特殊的细胞特性已成为细胞替 代治疗理想的种源细胞。 然而一方面包括人在内的灵长类动物的正常成体肝来 源的HPCs 的分离依然是很困难的；另一方面，ES 细胞来源的肝细胞和胆管细胞 的生成效率依旧很低。因此有必要建立稳定的高效的灵长类动物HPCs 细胞分离 培养体系及ES 细胞的肝细胞或胆管细胞分化体系以满足供体细胞的不足；这种 体系的建立还有利于研究肝细胞生物学如分化机制、自我更新机制等方面的重要 基础问题。 本研究以猕猴为实验模型，研究了正常成体肝来源的猕猴HPCs 分离、纯化 的条件，系统地鉴定了猕猴HPCs 的细胞特性和体内、外分化潜能，并评价了体 内移植效果。 同时以rES 为材料，建立了rES 高效分化为限定性内胚层 （definitive endoderm cells, DE）和胆管上皮细胞的分化体系。主要实验结 果包括：1）： FBS、EGF、HGF 及rat tail collagen (鼠尾胶原)是分离培养正 常成体猕猴来源的肝上皮前体细胞(rhesus monkey liver epithelial progenitor cells, mLEPCs)所必需的，mLEPCs 在此培养体系中至少可以扩增20 代或5 个月以上，并仍然保持原有的细胞特性；mLEPCs 呈现典型的上皮细胞形 态，并表达HPCs 细胞特有的表达模式即同时表达肝细胞和胆管细胞相关基因 （ALB，APOH，CX43，IB4）或蛋白（CK7，CK8，CK18）；在适宜的分化体系下， mLEPCs 可分化为功能性的肝细胞，形成具有胆管上皮细胞的胆管样结构，并能 转分化形成肌肉样细胞、肌样成纤维细胞及少突样细胞；移植入肝损伤的免疫抑 制的小鼠体内后，mLEPCs 能参与受体肝组织的再生，并能分化成ALB 阳性的肝 细胞；体内定位发现mLEPCs 与胆管区的细胞有相似的免疫原性，提示mLEPCs 可能来源于胆管区。2）：rES 在高浓度的acitvin A（100ng/ml）和低浓度的血 清(1%)单层诱导体系下可定向分化得到高比率的限定性内胚层细胞（definitive endoderm cells，DE 细胞）(约80%)； 高比率的DE 细胞的得到还与rES 细胞的接种密度相关；BMP4 和FGF1 可诱导DE 细胞高效向胆管上皮细胞分化（约90%）， 但并不能得到肝细胞；而Notch 信号通路可维持DE 细胞的存活，并决定着DE 细胞向胆管细胞分化，在Notch 信号通路失活的情形下，即使存在BMP4 和FGF1 都不能促使DE 细胞向胆管细胞分化。 本实验首次成功建立了正常猕猴成体肝HPCs 分离培养体系，证实了分离得 到的猕猴肝上皮前体细胞不但具有正常HPCs 的增殖活力和参与受体肝组织的再 生能力，而且还具有三个胚层的分化潜能，这一结果将为以HPCs 为基础的细胞 替代治疗人类肝脏疾病的实现提供了可能，并首次证明了HPCs 也可以像某些少 数成体干细胞一样具有三个胚层得分化潜能。 此外，本研究建立了rES 高效定 向分化为DE 细胞和胆管细胞的分化体系，这一方法的建立将促进灵长类动物的 DE 细胞的发育机制研究，同时也可为高比率的内胚层功能细胞（如胰岛细胞、 肝细胞、肺细胞）的获得提供丰富的种源细胞和平台。

人胚胎干细胞定向分化为神经前体细胞

人胚胎干细胞(human embryonic stem cells, hES细胞)来源于植入前胚胎的内细胞团，具有自我更新能力和发育全能性，能够在体内外分化为代表三个胚层的细胞类型。hES细胞来源的神经前体细胞（neural progenitor）对于研究胚胎早期的神经发育以及药物筛选和神经系统疾病的细胞替代性治疗具有重要意义。然而，许多因素影响了ES细胞的临床应用，如供体细胞不足、纯度低、异源物质污染等。 本研究采用同源饲养层培养的hES细胞在单层培养条件下高效地分化得到了神经前体细胞。主要结论如下：1）hES细胞在同源饲养层HAFi上培养八个月后仍保持ES细胞的各项表型特征和抗原特性。表明HAFi能够支持hES细胞的长期培养，从培养条件上避免了异源物质污染的可能性。2）单层贴壁分化的方法培养成分简单，不含血清和条件培养基，不需繁琐的筛选步骤就可以得到高比例的神经前体细胞（97.5%±0.83%）（P＜0.05）。此外，成分确定的培养基是研究神经分化的分子机制的良好模型。3）hES细胞来源的神经前体细胞具有分化为神经元，星形胶质和少突胶质细胞的能力，并能够模拟体内神经发育的过程和分子表达模式。长期的传代培养中发现，随着培养时间的延长，nestin阳性的神经前体细胞比例下降，同时发育能力也发生了变化。在传代培养的早期，神经前体细胞发育为神经元的比例很高，几乎没有胶质细胞分化出来。随培养时间的延长，胶质细胞的比例逐渐上升。进一步研究发现具有bHLH (basic helix-loop-helix) 结构域的转录因子neurogenein2(Ngn2) 和olig2可能在这一变化中发挥了重要的作用。

Ergot (Claviceps africana) ou doença açucarada do sorgo.

Morfologia da espigueta do sorgo; Suscetibilidade do progenitor feminino; Biologia do florescimento; Estadios da doenca; Formacao do estroma, Gota-de-mel, conidios e esclerocios; Ciclo da doenca e relacao patogeno-hospedeiro; Efeito dos exsudatos aderentes nas sementes; Disseminacao por insetos; Efeito de fertilizantes sobre a incidencia da doenca; Periodos latentes, de esporulacao e severidade; Identificacao de linhagens resistentes; Hospedeiros alternativos; Manejo integrado do Ergot.

Representações das crianças sobre a Casa de Abrigo e Sistema de Justiça

Dissertação apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Psicologia Jurídica

The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain

Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons

Isolation, characterization, and functional assessment of novel smooth muscle like stem progenitors

Vascular smooth muscle cells (VSMC) are one of the key players in the pathogenesis of cardiovascular diseases. The origin of neointimal VSMC has thus become a prime focus of research. VSMC originate from multiple progenitors cell types. In embryo the well-defined sources of VSMC include; neural crest cells, proepicardial cells and EPC. In adults, though progenitor cells from bone marrow (BM), circulation and tissues giving rise to SMC have been identified, no progress has been made in terms of isolating highly proliferative clonal population of adult stem cells with potential to differentiate into SMC. Smooth muscle like stem progenitor cells (SMSPC) were isolated from cardiopulmonary bypass filters of adult patients undergoing CABG. Rat SMSPC have previously been isolated by our group from the bone marrow of Fischer rats and also from the peripheral blood of monocrotaline induced pulmonary hypertension (MCT-PHTN) animal model. Characterization of novel SMSPC exhibited stem cell characteristics and machinery for differentiation into SMC. The expression of Isl-1 on SMSPC provided unique molecular identity to these circulating stem progenitor cells. The functional potential of SMSPC was determined by monitoring adoptive transfer of GFP+ SMSPC in rodent models of vascular injury; carotid injury and MCT-PHTN. The participation of SMSPC in vascular pathology was confirmed by quantifying the peripheral blood, and engrafted levels of SMSPC using RT-PCR. In terms of translating into clinical practice, SMSPC could be a good tool for detecting the atherosclerotic plaque burden. The current study demonstrates the existence of novel adult stem progenitor cells in circulation, with the potential role in vascular pathology.

Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo.

Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. Here, we provide a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examine the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. We observed that endomesoderm segregation first occurs at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. Our experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate.

Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.

The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.

Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens), This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions, pLR was synthesized and elicited rapid, noncytolytic histamine release that had a a-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin, pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action, pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i,e. mature neutrophils, pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

High-resolution Keck I spectroscopy of Galactic halo post-asymptotic giant branch stars

Absolute and differential abundance analyses have been performed from high-resolution, high signal-to-noise ratio optical (Keck I) spectra for three evolved Galactic halo stars, namely PG 1704 + 222, HD 341617 and LSIV -0401. Their derived atmospheric parameters indicate that all three objects are undergoing a post-asymptotic giant branch (post-AGB) phase of evolution. A differential abundance analysis reveals HD 341617 as having a mild carbon deficiency of 0.74 dex, possibly due to the star having evolved off the AGB before the onset of the third dredge-up. Although such carbon underabundances are typical of hot post-AGB objects, the same trend is not observed in PG 1704 + 222, where the carbon abundance is found to be consistent with those derived for nitrogen and oxygen. Hence, a dredge-up scenario need not be invoked to explain the chemical composition of PG 1704 + 222. For LSIV -0401 no iron deficiency is apparent relative to magnesium and silicon, and hence a gas- dust separation event in the AGB progenitor need not be invoked for this star.